Related Viroids Research Articles

Reassessing species demarcation criteria in viroid taxonomy by pairwise identity matrices.

With a small, circular and non-protein coding RNA genome, viroids are the smallest infectious agents. They invade plants, which in turn may develop symptoms. Since their discovery about 50 years ago, more than thirty viroids have been reported and classified using as species demarcation less than 90 per cent sequence identity on the overall genome and evidence of biological divergence with respect to the closest related viroids. In the last few years, new viroids have been identified that infect latently their (frequently) woody hosts and have a narrow experimental hosts range, complicating and slowing down studies on their biology. As a consequence, several viroids are still waiting for classification. Moreover, the number of new viroids is expected to increase in the next years due to the use of high-throughput sequencing technologies with diagnostics purposes. Therefore, establishment of reliable species demarcation criteria mainly based on molecular features of viroids is needed. Here, viroid classification is reassessed and a scheme based on pairwise sequence identity matrices is developed. After identifying a threshold pairwise identity score (PWIS) for each viroid genus, to be used as a species demarcation criterion, we show that most of those yet unclassified viroids can be assigned to a known or to a new species, thus limiting the need for additional biological evidence to only a few more complex situations. The advantages of this PWIS-based method are that the proposed identity thresholds for species demarcations are not arbitrarily established and evidence for biological divergence is not mandatory. Importantly, the current classification is not essentially modified. A protocol for a tentative fast classification of new viroids according to the proposed approach is also provided.

Evaluation of viroid extraction methods and application of a one-step reverse transcription real-time polymerase chain reaction assay (RT-qPCR) for the rapid detection of Chrysanthemum stunt viroid (CSVd) infection

Early viroid detection in chrysanthemum cultivars depends on both an efficient RNA extraction method as well as a sensitive detection assay. In this study, we evaluated RNA purification methods and optimized a one-step RT-qPCR assay for the detection of Chrysanthemum stunt viroid (CSVd). Three commercially available RNA extraction technologies (silica and silicon carbide spin column as well as the phenol/chloroform extraction) were compared for efficacy in CSVd detection sensitivity. We found a 10- to 50-fold improvement in CSVd detection sensitivity when using silicon carbide (SiC) spin columns to purify RNA compared with the silica-based column or the traditional phenol/chloroform extraction method. To optimize detection sensitivity and reduce detection time, a commercial CSVd end-point PCR primer set was adapted to a SYBR Green based one-step RT-qPCR assay. Standard curve analysis estimated that the limit of detection was about 54 CSVd copies and the specificity test against six related viroids showed no cross-reactivity. As a part of the validation, the one-step RT-qPCR detection system was compared with RT-PCR to detect CSVd in both randomly selected greenhouse plants and mechanically inoculated chrysanthemum varieties. Cultivars selected from a commercial greenhouse with >50% infection levels were tested for CSVd presence. Cultivars ‘Pelee’, ‘Puma’ and ‘Shamrock’ tested positive by both the one step RT-qPCR and RT-PCR but CSVd was not detected in ‘Icecap’ and ‘Snowball’. In mechanical inoculation studies, CSVd was detected in 5 of 6 chrysanthemum cultivars – ‘Chesapeake’, ‘Durango’, ‘Juneau’, ‘Pelee’ and ‘Viron’ but not in ‘Pueblo’. No differences in detection were evident between the RT-qPCR and RT-PCR methods.

Rapid in vitro microindexing of viroids in citrus

A new laboratory technique combining shoot‐tip grafting in vitro and biological indexing on indicator plants was explored for the detection of citrus exocortis and related viroids. Τhree in vitro laboratory methods were used and compared with the classical biological method. With the classical in vivo method, diagnosis is based on the expression of symptoms on indicators 11–14 weeks after inoculation. In contrast, with the first in vitro method, microindexing in vitro of citron seedlings by graft inoculation, diagnosis was possible 12 days after inoculation; with the second method, microindexing in vitro of citron cuttings by graft inoculation, 20 days after inoculation; and with the third method, microindexing in vitro of citron cuttings by injection inoculation, 40 days after inoculation. Inoculated Etrog citron plantlets grown in vitro and tested by RT‐PCR showed the same viroid content as the source plants. Of the three in vitro viroid indexing methods, microindexing on cuttings by grafting was easier and more reliable than microindexing either on seedlings or on cuttings by injection.

Potato spindle tuber viroid: the simplicity paradox resolved?

Potato spindle tuber viroid (PSTVd) is the type species of the genus Posipiviroid, family Pospiviroidae. An absence of hammerhead ribozymes and the presence of a 'central conserved region' distinguish PSTVd and related viroids from members of a second viroid family, the Avsunviroidae. Viroids are small, unencapsidated, circular, single-stranded RNA molecules which replicate autonomously when inoculated into host plants. Because viroids are non-protein-coding RNAs, designation of the more abundant, highly infectious polarity strand as the positive strand is arbitrary. PSTVd assumes a rod-like, highly structured conformation that is resistant to nuclease degradation in vitro. Naturally occurring sequence variants of PSTVd range in size from 356 to 361 nt. HOSTS AND SYMPTOMS: The natural host range of PSTVd-cultivated potato, certain other Solanum spp., and avocado-appears to be quite limited. Foliar symptoms in potato are often obscure, and the severity of tuber symptoms (elongation with the appearance of prominent bud scales/eyebrows and growth cracks) depends on both temperature and length of infection. PSTVd has a broad experimental host range, especially among solanaceous species, and strains are classified as mild, intermediate or severe based upon the symptoms observed in sensitive tomato cultivars. These symptoms include shortening of internodes, petioles and mid-ribs, severe epinasty and wrinkling of the leaves, and necrosis of mid-ribs, petioles and stems.

Precisely full length, circularizable, complementary RNA: an infectious form of potato spindle tuber viroid.

The replication of many viral and subviral pathogens as well as the amplification of certain cellular genes proceeds via a rolling circle mechanism. For potato spindle tuber (PSTVd) and related viroids, the possible role of a circular (-)strand RNA as a template for synthesis of (+)strand progeny is unclear. Infected plants appear to contain only multimeric linear (-)strand RNAs, and attempts to initiate infection with multimeric (-)PSTVd RNAs generally have failed. To examine critically the infectivity of monomeric (-)strand viroid RNAs, we have developed a ribozyme-based expression system for the production of precisely full length (-)strand RNAs whose termini are capable of undergoing facile circularization in vitro. Mechanical inoculation of tomato seedlings with electrophoretically purified (-)PSTVd RNA led to a small fraction of plants becoming infected whereas parallel assays with an analogous tomato planta macho viroid (-)RNA resulted in a much larger fraction of infected plants. Ribozyme-mediated production of (-)PSTVd RNA in transgenic plants led to the appearance of monomeric circular (-)PSTVd RNA and large amounts of (+)PSTVd progeny. No monomeric circular (-)PSTVd RNA could be detected in naturally infected plants by using either ribonuclease protection or electrophoresis under partially denaturing conditions. Although not a component of the normal replicative pathway, precisely full length (-)PSTVd RNA appears to contain all of the structural and regulatory elements necessary for initiation of viroid replication.

Occurrence of Peach Latent Mosaic Viroid (PLMVd) on Plum in Italy.

Peach latent mosaic disease was originally described in France in 1976 (1). The disease is often latent in peach trees but may induce mosaic symptoms on leaves, irregularly shaped colorless fruit with cracked sutures and enlarged pits, bud necrosis, and delay in foliation, flowering, and fruit maturity. Recently, the disease has been shown to be caused by peach latent mosaic viroid (PLMVd) (2). PLMVd is distributed worldwide on peach. A recent survey in Italian plum orchards has showed the occurrence of PLMVd in two plum cultivars (Black Diamond and Angeleno) at different geographical locations in central Italy. Plum samples showed bark necrosis and bark split on branches and trunk; moreover, 1-or 2-year-old shoots and stem appeared dwarfed because of shortening of internodes and reduced growth. Budwood from plum samples were grafted, in greenhouse, onto peach GF305 indicator plants. Total nucleic acids (TNA) were then extracted from peach GF 305, or plum leaves, after 6 months of postinoculation. The presence or absence of PLMVd was ascertained by bidirectional polyacrylamide gel electrophoretic (dPAGE) analysis and by spot hybridization assay with PLMVd cRNA probe provided by A. Hadidi. A dPAGE analysis of TNA extracted from grafted peach GF305 showed a band with the same electrophoretic mobility as that of D-168 strain of PLMVd positive control. No bands were observed in healthy peach GF305 or from healthy or diseased plum extracts. The lack of detection of a viroid band from diseased plum samples by dPAGE analysis may be due to low titer of the viroid in infected plum. Molecular hybridization analysis of total nucleic acids with PLMVd cRNA probe, however, showed positive hybridization signals with nucleic acids from grafted peach GF305 or infected plum. Thus, PLMVd or a very closely related viroid infects plum trees. Moreover, on the basis of biological and serological assays, apple chlorotic leaf spot trichovirus, in a sample, and prunus necrotic ring spot ilarvirus, in the second one, were found in association with PLMVd. The same symptoms observed in the two PLMVd-infected plums occurred in other plants infected only by the two viruses (alone or in association). These data suggest that, very probably, PLMVd does not play a specific role in the etiology of the field symptomatology described above. In any case, this is the first report for the occurrence of PLMVd on plum in Italy.

Occurrence of Peach Latent Mosaic Viroid in Stone Fruits and Its Transmission with Contaminated Blades.

Peach latent mosaic viroid (PLMVd) is widely distributed (approximately 55%) in peach germplasm from Europe, Asia, North America, and South America. PLMVd, or a closely related viroid, was occasionally detected in cherry, plum, and apricot germplasm from countries in Europe or Asia. The cherry isolate of PLMVd is 337 nucleotides in length and is 91 to 92% homologous to PLMVd isolates from peach. Molecular hybridization experiments demonstrated that PLMVd is not related to the agent of peach mosaic disease. PLMVd was readily transmitted (50 to 70%) by contaminated blades to green shoots and lignified stems of peach GF-305 plants. These results indicate that the viroid may be transmitted in orchards with contaminated pruning equipment.

Identification and molecular properties of a 306 nucleotide viroid associated with apple dimple fruit disease.

A new viroid associated with an apple fruit disorder similar to, but more severe than, the dapple apple disease induced in some varieties by apple scar skin viroid (ASSVd) has been found. The new viroid, tentatively termed apple dimple fruit viroid (ADFVd), is a circular RNA of 306 nucleotides which adopts a quasi-rod-like conformation of minimum free energy. It contains the core nucleotides of the central conserved region (CCR) of the ASSVd group as well as the terminal conserved region (TCR) present in all members of the ASSVd and potato spindle tuber viroid (PSTVd) monophyletic groups. ADFVd has the highest sequence similarity with ASSVd and the 294 nucleotide citrus viroid CVd-IIIb sharing with the latter an almost identical left terminal domain. The right- and left-hand termini of ADFVd are formed by almost perfect duplications of sequences found in the CCR upper and lower strands, respectively, of PSTVd and closely related viroids.

Replication of avocado sunblotch viroid: evidence for a symmetric pathway with two rolling circles and hammerhead ribozyme processing.

The structure of a series of RNAs extracted from avocado infected by the 247-nt avocado sunblotch viroid (ASBVd) was investigated. The identification of multistranded complexes containing circular ASBVd RNAs of (+) and (-) polarity suggests that replication of ASBVd proceeds through a symmetric pathway with two rolling circles where these two circular RNAs are the templates. This is in contrast to the replication of potato spindle tuber viroid and probably of most of its related viroids, which proceeds via an asymmetric pathway where circular (+)-strand and linear multimeric (-)-strand RNAs are the two templates. Linear (+) and (-) ASBVd RNAs of subgenomic length (137 nt and about 148 nt, respectively) and one linear (+)-strand ASBVd RNA of supragenomic length (383-384 nt) were also found in viroid-infected tissue. The two linear (+)-strand RNAs have the same 5'- and 3'-terminal sequences, with the supragenomic species being a fusion product of the monomeric and subgenomic (+)-strand ASBVd RNAs. The 3' termini of these two (+)-strand molecules, which at least in the subgenomic RNA has an extra nontemplate cytidylate residue, could represent sites of either premature termination of the (+)-strands or specific initiation of the (-)-strands. The 5' termini of sub- and supragenomic (+)-strand and the 5' terminus of the subgenomic (-)-strand ASBVd RNA are identical to those produced in the in vitro self-cleavage reactions of (+) and (-) dimeric ASBVd RNAs, respectively. These observations strongly suggest that the hammerhead structures which mediate the in vitro self-cleavage reactions are also operative in vivo.

Detection of avocado sunblotch viroid in chloroplasts of avocado leaves by in situ hybridization.

In situ hybridization experiments were carried out to detect avocado sunblotch viroid (ASBVd) in foliar tissue of avocado, using a digoxigenin-labelled RNA probe complementary to the ASBVd-RNA in sections of aldehyde-fixed, LRGold-embedded leaf samples. Detection of the probe was made through anti-digoxigenin antibody and protein-A colloidal gold (20 nm). Seventy to 80% of the signals came from chloroplast while the cytoplasm and vacuole were labelled with ca. 10% of the gold particles. This is in contrast with the subcellular localization of potato spindle tuber viroid and some other related viroids, which are mainly found in the nucleus.

Effects of random mutagenesis upon potato spindle tuber viroid replication and symptom expression

A combination of random chemical mutagenesis plus temperature gradient gel electrophoresis was used to isolate a collection of 57 potato spindle tuber viroid (PSTV) cDNAs containing mutations distributed throughout the entire 359 nucleotide genome. Although the presence of multiple mutations was often associated with a loss of cDNA infectivity, infectious PSTV cDNAs containing as many as four unlinked alterations could be isolated. Several mutations in the pathogenicity domain and left terminal loop were stably maintained in the resulting progeny, but those which affect base pairing in secondary hairpins I and II were either lethal or rapidly reverted to wild-type. One stable C → U substitution which may promote significant structural rearrangement within the right side of the pathogenicity domain had no detectable effect upon symptom expression. The variable domains of several noninfectious mutants contained an A → G substitution which is likely to inhibit the in vitro formation of secondary hairpin II via stabilization of the native structure, and the lethal nature of this mutation was confirmed by oligonucleotide-directed mutagenesis. Several lines of evidence now point toward an essential role for secondary hairpin II in the replication of PSTV and related viroids.

Coconut Cadang-Cadang Disease and Its Viroid Agent

In the early 1930s a devastating epidemic of a lethal disease of coconut palm (Cocos nucijera L.) was reported from southern Luzon in the Philippines. It was appropriately named disease from the word gadan-gadan of the local dialect meaning dying (13). Anecdotal reports indicate that the disease probably occurred a few years earlier in the Philippines, at about the time a somewhat similar epidemic was reported on the island of Guam. The disease in Guam was called and was extremely destructive to the island's coconut population (2). Both diseases have now been shown to be caused by related viroids: coconut cadang-cadang viroid (CCCVd) and coconut tinangaja viroid (CTiVd).

Two related viroids cause grapevine yellow speckle disease independently.

We have confirmed that two closely related circular RNA molecules previously named grapevine yellow speckel viroid (GYSV) and grapevine viroid 1B (GV1B) are indeed viroids. Electron microscopy after spreading under non-denaturing conditions revealed that GYSV has a rod-like structure typical of viroids. Purified GYSV and GV1B replicated independently in inoculated grapevine seedlings and some of the infected plants developed yellow speckle symptoms indicating that both viroids can cause grapevine yellow speckle disease. Plus-sense RNA transcripts derived from a dimeric GYSV cDNA clone induced yellow speckle symptoms in a grapevine seedling confirming the role of GYSV in the yellow speckle disease. Two oligonucleotide probes were synthesized for the detection of the two related viroids. The probes which could detect each viroid individually were used to assess correlations between the occurrence of these viroids and the incidence of the disease.

Self-cleavage of rna in the replication of viroids and virusoids

Viroids are infectious, circular RNA molecules of 246 to 375 nucleotides found in plants. Virusoids are of similar size and structure but they are dependent on, and encapsidated in, a helper virus. A rolling circle mechanism of replication is considered to account for the presence of greater-than-unit-length plus and minus RNAs of both viroids and virusoids found in infected plants. An essential feature of this mechanism is the specific processing or cleavage of high molecular weight intermediates to produce linear monomers which are then ligated to circular monomers. We have investigated the putative processing cleavage reactions using in vitro-synthesized RNA transcripts of dimeric cDNA clones of the 247-nucleotide avocado sunblotch viroid (ASBV) and of partial cDNA clones of the 324-nucleotide virusoid of lucerne transient streak virus (vLTSV). In both cases, there is a specific, non-enzymic, self-cleavage of plus as well as minus transcripts. The plus and minus sites of cleavage are in neighbouring parts of ASBV and of vLTSV and highly conserved two-dimensional structures can be drawn around the cleavage sites as well as around the putative or demonstrated cleavage sites of precursors of the virusoids of three other viruses and of the linear satellite RNA of tobacco ringspot virus. The results also indicate that the sole function of about one-third of the ASBV and vLTSV molecules is provision of sequences that allow the formation of the self-cleavage structures of both 'plus' and 'minus' RNA precursors during the replication cycle. Similar self-cleavage of 'plus' RNA transcripts of a dimeric cDNA clone of citrus exocortis virus (CEV) was not observed. However, the putative processing site for CEV precursors was located within three nucleotides by site-directed mutagenesis. No two-dimensional structures similar to those found for ASBV and vLTSV were found around the processing site. It is possible that a different type of self-cleavage or enzymic processing event occurs during the replication cycle of CEV and related viroids.

Spot hybridization: Application to viroid identification

Summary A cloned 32 P-labelled cDNA probe to potato spindle tuber viroid (PSTV) was used to investigate the possibility of applying the spot hybridization technique to the identification of 2 virois PSTV and chrysanthemum stunt viroid (CSV). The best conditions for obtaining the highest sensitivity consisted of spotting heat-denatured samples onto the nitro-cellulose membrane, then hybridizing at 42 or 55°C in the presence of 50% formamide. The technique led to detection of 100–250 pg of purified viroid RNA and to identification of the viroid in a plant extract corresponding to 0.1 mg of infected leaves. These values demonstrated a clear advantage of the hybridization technique over the electrophoretic assay. A 3 H-labelled probe could be substituted for the 32 P probe, but it exhibited poor sensitivity due to the initially low specific radioactivity of the 3 H probe. Despite large sequence homologies, the PSTV 32 P-labelled probe hybridized only with CSV in purified solutions under non-stringent conditions, e. g. low temperature (42°C) and in the presence of relatively high amounts of CSV (50 mg). Therefore, spot hybridization appeared to be a more sensitive diagnostic technique than the electrophoretic assay, but with a high degree of specificity even with apparently closely related viroids.