The question of Citrus viroid IV as a cocadviroid.

It has been suggested that Citrus viroid IV (CVd-IV) be classified as a species within the genus Cocadviroid. This relationship was based on the presence of a terminal conserved hairpin (TCH) and absence of a terminal conserved region (TCR) as specific structural motifs in common with isolates of Coconut cadang-cadang viroid (CCCVd) as well as phylogenetic relationships with members of the genus Cocadviroid. Evidence is presented for a "vestigial" TCR in CVd-IV as well as the introduction of the terminal repeat region (TRR) motif and an alternative sequence analysis that suggests a closer phylogenetic relationship of CVd-IV to isolates of Citrus exocortis viroid (CEVd), a species within the genus Pospiviroid than to CCCVd. This position is further supported by the striking similarity of biological properties between CVd-IV and CEVd with the suggestion offered that biological evidence be considered for specific adjustments to any overall classification scheme for viroids.

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.

RNA silencing as related to viroid induced symptom expression.

Evidence of post-transcriptional gene silencing (PTGS) in avocado infected by Avocado sunblotch viroid (ASBVd), the type species of family Avsunviroidae, was suggested by detection of ASBVd-specific 22-nucleotide RNAs. PTGS was observed in infected bleached and variegated symptomatic tissues as well as symptomless carrier foliar sources and fruit with typical sunblotch disease lesions. Tissues with the different symptom expressions, characterized by the presence of different predominant ASBVd variants, were found to induce PTGS at differential levels. Detection of the PTGS-associated small interfering RNAs (siRNAs) as well as relative concentration was also related to viroid titer. PTGS induced in Gynura aurantiaca infected with two closely-related variants of Citrus exocortis viroid, a member of family Pospiviroidae, was not directly related to viroid titer with initiation of symptoms.

Finger Imprint of Poncirus trifoliata: A Specific Interaction of a Viroid, a Host, and Irrigation.

The unusual symptom, "finger imprint", described exclusively on Poncirus trifoliata, has been reported in only a single field trial investigating the effects of citrus viroids on crop performance. With this, the question has persisted whether the observed growth abnormality was a disease symptom induced by Citrus viroid IIIb (CVd-IIIb) or a consequence of mechanical damage caused by the handling of young trees during propagation or cultural practices in the field. The recurrence of finger imprint symptoms on trees after 5 years in the field in which no abnormal growth features were previously noted now supports the proposition of a viroid-induced disease. The symptom expression results from an unusual etiology of a complex relationship of the specific viroid CVd-IIIb on the specific rootstock P. trifoliata only when supplemental water is applied by sprinkler irrigation.

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.

Correlation of hop stunt viroid variants to cachexia and xyloporosis diseases of citrus.

ABSTRACT Citrus viroid (CVd) group II is comprised of hop stunt viroid (HSVd)-related variants of 295 to 302 nucleotides. Included in this group are the cachexia-inducing agents citrus cachexia viroid (or CVd-IIb), CVd-IIc, Ca-903, and Ca-909 as well as the non-cachexia-inducing variant CVd-IIa. The cachexia indexing hosts 'Parson's Special' mandarin and 'Orlando' tangelo as well as Citrus macrophylla responded with symptoms of gumming, discoloration, and stem pitting when infected by CVd-IIb, CVd-IIc, or Ca-903. However, 'Palestine' sweet lime, the indicator host used to describe the xyloporosis disease, displayed a distinctly different fine-pitting reaction and no discoloration or gumming when infected by the same viroids. Cachexia-inducing variants contain a number of nucleotide changes more similar to hop-type HSVd sequences than to the citrus-type HSVd sequences, as typified by CVd-IIa. The nucleotide sequence of CVd-IIc was identical to CVd group II isolates common to trees expressing xyloporosis. Experimental evidence indicates that either CVd-IIb or CVd-IIc can cause citrus diseases known as cachexia and xyloporosis and that the two disease designations reflect the distinct responses of different indexing hosts to the same viroids.

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.

Regulation of pathogenicity in hop stunt viroid-related group II citrus viroids.

Nucleotide sequences were determined for two hop stunt viroid-related Group II citrus viroids characterized as either a cachexia disease non-pathogenic variant (CVd-IIa) or a pathogenic variant (CVd-IIb). Sequence identity between the two variants of 95.6% indicated a conserved genome with the principal region of nucleotide difference clustered in the variable (V) domain. Full-length viroid RT-PCR cDNA products were cloned into plasmid SP72. Viroid cDNA clones as well as derived RNA transcripts were transmissible to citron (Citrus medica L.) and Luffa aegyptiaca Mill. To determine the locus of cachexia pathogenicity as well as symptom expression in Luffa, chimeric viroid cDNA clones were constructed from segments of either the left terminal, pathogenic and conserved (T1-P-C) domains or the conserved, variable and right terminal (C-V-T2) domains of CVd-IIa or CVd-IIb in reciprocal exchanges. Symptoms induced by the various chimeric constructs on the two bioassay hosts reflected the differential response observed with CVd-IIa and -IIb. Constructs with the C-V-T2 domains region from clone-IIa induced severe symptoms on Luffa typical of CVd-IIa, but were non-symptomatic on mandarin as a bioassay host for the cachexia disease. Constructs with the same region (C-V-T2) from the clone-IIb genome induced only mild symptoms on Luffa, but produced a severe reaction on mandarin, as observed for CVd-IIb. Specific site-directed mutations were introduced into the V domain of the CVd-IIa clone to construct viroid cDNA clones with either partial or complete conversions to the CVd-IIb sequence. With the introduction of six site-specific changes into the V domain of the clone-IIa genome, cachexia pathogenicity was acquired as well as a moderation of severe symptoms on Luffa.

A citrus exocortis viroid variant from broad bean (Vicia faba L.): infectivity and pathogenesis.

A viroid present in very low titres was isolated from symptomless field broad bean plants. It was identified as a variant of citrus exocortis viroid in the T2, V and C domains. Infection of several hosts resulted in a change in the composition of the viroid population. Serial passage through tomato and back to the host of origin, broad bean, resulted in major changes in replication efficiency, host range and pathogenicity. The unique nucleotide sequence differences identified in the original broad bean variant were not conserved after passage through alternative hosts. The effects of these sequence variations on viroid secondary structure result in nonpathogenic viroid variants which can remain unnoticed in certain plant species but may act as reservoirs of viroid disease.

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.

Separation of viroid RNAs by cellulose chromatography indicating conformational distinctions.

A series of viroids and viroid-like RNAs extracted from citron (Citrus medica), grapevine (Vitis vinifera), and avocado (Persea americanum) displayed differential binding capacities to cellulose in the presence of ethanol. This nonionic interaction was influenced by the presence of magnesium ions suggesting variations in conformation among the viroid RNAs and corresponding differences in reactivity with cellulose. The specific elution profiles present a basis for the subclassification of groups of viroid-like molecules.

Enhanced resolution of circular and linear molecular forms of viroid and viroid-like RNA by electrophoresis in a discontinuous-pH system.

A discontinuous-pH polyacrylamide gel electrophoresis system is described. An increase in the pH differential between the gel and the running buffer enhances the separation of low molecular weight circular and linear RNA molecules. Highly purified preparations of the circular form of viroids can be obtained with this procedure. Since all the linear RNAs of similar molecular weight migrate with the front, a relatively clean background can be obtained even when crude extracts are used. This facilitates an improved separation and identification of similarly sized viroid-like RNAs. The conditions of electrophoresis in low salt and 8 M urea also permit the effective transfer of RNA molecules directly to nylon-based membranes without any additional denaturation treatment.

Characterization of viroid-like RNAs associated with the citrus exocortis syndrome.

Three additional viroid-like RNA species (RNA-I, -II, and -III), smaller than the 371-nucleotide citrus exocortis viroid (CEV), have been identified in citron (Citrus medica) trees which display symptoms of the exocortis disease. The three RNAs migrate on polyacrylamide gels under denaturing conditions in the region between CEV and avocado sunblotch viroid circular molecules, indicating a size range of about 311-335 nucleotide residues in both circular and linear molecular forms. RNA-II is removed from the preparations when chromatographed on CF-11 cellulose. All three RNAs fail to replicate when introduced into Gynura aurantiaca, a herbaceous host of CEV, yet RNA-I and RNA-III can be independently transmitted to citron. Dot-blot hybridization with CEV-cDNA indicates no significant homology between CEV and RNA-I, RNA-II, or RNA-III. The relationship among CEV-RNA and the three viroid-like RNA species in the segregation of expression and intensity of the exocortis disease reaction is discussed.

Optimal conditions for cell-free synthesis of citrus exocortis viroid and the question of specificity of RNA polymerase activity.

Cell-free synthesis of citrus exocortis viroid (CEV) in nuclei-rich preparations from infected Gynura aurantiaca was optimum at 18-24 degrees C. Incubation of reaction mixtures at higher temperatures (30-36 degrees C) resulted in an increase of CEV linear molecules and the recovery of incomplete or nicked newly synthesized RNA species. Although the Mg(2+) optimum (2.5-5 mM) for CEV synthesis was lower than that for total [(32)P]CMP incorporation (10 mM), the response to Mn(2+) ion was distinctly different. Whereas maximum total activity was observed in 1 mM Mn(2+) with a pronounced reduction (80%) in 5 mM Mn(2+), CEV synthesis was maintained in 1-15 mM Mn(2+). Inhibition of alpha-amanitin-sensitive CEV synthesis in 200 mM (NH(4))(2)SO(4) resembles the reaction of RNA polymerase II on a free nucleic acid template. However, detection of trace levels of alpha-amanitin-resistant CEV synthesis activity inhibited by low (NH(4))(2)SO(4) concentrations (25 mM) suggests the possible involvement of RNA polymerase I- and/or III-like activity.

Enhanced detection of viroid-RNA after selective divalent cation fractionation.

Treatment of nucleic acid preparations from citrus exocortis viroid infected tissues with Ca2+, Co2+, Mn2+, and Zn2+ results in rapid precipitation of all large RNA molecules and selective precipitation of low-molecular-weight RNA species. Analysis of the viroid-RNA by polyacrylamide gel electrophoresis is greatly enhanced in the absence of any additional treatment with 2 M LiCl. The ratio of circular to linear molecules as well as the relative infectivity of Mn2+ -treated viroid preparations remain unaffected.