Host-directed processing of Citrus exocortis viroid.

Prolonged infection of tomato hybrid (Lycopersicon esculentum x Lycopersicon peruvianum) by Citrus exocortis viroid (CEVd) resulted in viroid-like enlarged structures, detected by gel electrophoresis. This population included two new enlarged variants or D-variants, D-87 and D-76, and three transient species or D-forms, D-38, D-40 and D-43. Sequence analyses exposed a locus near the terminal repeat region where major changes appeared consistently. In transmission tests to CEVd hosts, a variety of progeny populations were recovered, including progeny enlargements of and reversions to CEVd, as well as sequence fidelity to the inoculum. Transmission tests to citrus hosts of the genera Citrus, Poncirus or Fortunella were unsuccessful. The importance of host specificity to the recovery and processing of the various CEVd-related structures, as well as the temporal variability of progeny populations, was demonstrated.

CEVd-induced symptom modification as a response to a host-specific temperature-sensitive reaction.

Natural selection of two new variants of citrus exocortis viroid (CEVd) was detected by observing tissues displaying both severe and mild symptoms from a single Gynura aurantiaca. The variants CEVd-S (severe) and CEVd-M (mild), differing by only five nucleotides confined to the pathogenic (P) domain, remained stable when propagated by rooted cuttings or from successive plants inoculated with tissue extracts or transcripts from cDNA clones. CEVd-S induces a very severe reaction in Gynura that is consistent throughout a range of environmental conditions. However, symptoms resulting from CEVd-M infection can vary from a nonsymptomatic condition to a severe reaction when grown at 40 degrees C. This differential response was confined to a single host, Gynura aurantiaca, and expressed under standard growing conditions. The distinct host responses induced by these variants could not be correlated with any changes in sequence or conformation of the dominant viroid variant, as predicted by molecular modeling. Therefore, the variable symptom expression appears to be associated with a specific temperature-sensitive response of Gynura aurantiaca.

Population diversity in grapevine yellow speckle viroid-1 and the relationship to disease expression.

Vitis vinifera cultivars Zinfandel-1A and Mission were found to harbor different grapevine yellow speckle viroid-1 (GYSVd-1) variants and characterized to define the relationship to yellow speckle (YS) and vein-banding (VB) diseases. Products from the left terminal (T1), pathogenic (P), and a portion of the central (C) domains of Zinfandel-1A and Mission displayed distinct single-stranded conformation polymorphism (SSCP) patterns, presumably reflecting nucleotide changes in the P domain. The two selections were shown to contain homogeneous populations of type 1 and type 2 GYSVd-1 variants described in Australia. Symptoms of YS were induced only in vines containing the type 2 variant by treatment at a constant temperature of 32 degreesC in continuous light. SSCP of Pagadebit selections from Italy revealed the nonsymptomatic variant was essentially identical to Zinfandel-1A, whereas symptomatic selections were unlike any other previously described. Nucleotide sequence confirmed that nonsymptomatic selections from Italy contained the GYSVd-1 type 1 variant. A total of 43 changes were spread throughout the T1, C, V, and T2 domains from symptomatic selections. This study establishes the Australian type 1 variant as the non-symptom-inducing form of GYSVd-1 and type 2 as the symptom-inducing variant. The distinct symptom-inducing variant from Italy is proposed as a new type 3 variant of GYSVd-1.

Structural sites specific to citrus viroid groups.

Synthesis of cDNA probes by random-priming of a viroid template displays the unusual property of specificity to all members included within a single citrus viroid Group. The specificity of hybridization reactions was influenced by the structural conformation of the viroid RNA template, reaction conditions for reverse transcription and hybridization protocols. Mapping the loci for probe transcription from the CEVd, CVd-IIb, and CVd-IV genomes suggests that a similar structured conformation may be responsible for group specificity. A stem-loop configuration in the viroid template and hybridization target sites can be proposed to be responsible for the availability of the group-specific sequences.

Nucleotide sequence and structural features of the group III citrus viroids.

The nucleotide sequence and secondary structure of two representative variants from the Group III citrus viroids. CVd-IIIa (297 bases) and CVd-IIIb (294 bases) were determined. The variants are related to the apple scar skin viroid (ASSVd) family. Although smaller in size than any of the ASSVd-related viroids, the central conserved region as well as most of the terminal conserved region of ASSVd is retained. The rod-like structural configuration (characteristic of ASSVd) of the variants as predicted by minimum free energy analysis is presented.

Avocado sunblotch disease: a persistent viroid infection in which variants are associated with differential symptoms.

Variants of avocado sunblotch viroid (ASBVd) of between 247 and 250 nucleotides in length have been recovered from diseased avocado tissues. The sunblotch syndrome covers a complex pattern of disease symptoms which are associated with infection by variants of ASBVd. The viroid species are designated ASBVd-B, ASBVd-V and ASBVd-Sc from their association with bleached, variegated or symptomless carrier tissues respectively. Host-viroid interactions and structural relationships among the variants suggest a transition in sunblotch disease from a severe acute to a persistent mild form of infection.

A stable 463 nucleotide variant of citrus exocortis viroid produced by terminal repeats.

An unusual variant of citrus exocortis viroid (CEV) was detected when an inoculum source from Gynura aurantiaca D.C. was used to infect a hybrid tomato (Lycopersicon esculentum Mill. x L. peruvianum). The 92 nucleotide larger variant, CEV D-92, which displayed the characteristic circular and linear viroid structural forms, contained two repeated sequences spanning the V and T2 domains. A dramatic moderation of symptom expression in Gynura accompanied the incorporation of these repeated sequences. A comparison of the sequence and structure of CEV D-92 with coconut cadang-cadang viroid revealed similarities in the regions generating the naturally occurring terminal repeats suggesting a possible preferred site for RNA recombination between viroids.

Isolates of citrus exocortis viroid recovered by host and tissue selection.

Isolates of citrus exocortis viroid (CEV) from a single sweet orange citrus source have been selected by sequential passage through the alternative hosts citron, Gynura aurantiaca, a hybrid tomato Lycopersicon esculentum x L. peruvianum, and from disorganized callus culture of the hybrid tomato. The distinctions in symptom expression, titre and electrophoretic mobility among the CEV isolates, operationally termed CEVc (citron), CEVg (Gynura), CEVt (tomato) and CEVcls (callus) are supported by characteristically different nucleotide sequences. The nucleotide sequence of full-length cDNA clones of CEVc purified from citron shows exchanges not reported for any previously described CEV variant. An unusual number of exchanges have been localized in the terminal domains of all the isolates analysed here. A common pattern of nucleotide exchanges, described as a 'tomato signature', can be detected in all of the isolates derived from hybrid tomato tissues.

Relationships among the viroids derived from grapevines.

There have been numerous reports of grapevine viroids, describing physical and biological properties suggestive of similar or identical molecular forms. With consideration of these properties and the application of random-primed and specific cDNA probes, four major groups of grapevine viroids have been defined. Designations which can be used to describe distinct viroids within the four groups include (i) CEVd-g, a grapevine isolate of citrus exocortis viroid, (ii) GVd-c, a grapevine viroid recovered from cucumber, and AGVd, Australian grapevine viroid, (iii) GYSVd-1 and GYSVd-2, two viroids inducing yellow speckle disease and (iv) HSVd-g, a grapevine isolate of hop stunt viroid.