Citrus Exocortis Viroid Isolates Research Articles

First report of Citrus bent leaf viroid in the United Arab Emirates.

Citrus species are among the three top fruit trees in the United Arab Emirates (UAE), with a combined total pro- duction of 12230 tons in 2011. Leaf samples were collected from four lemon (Citrus lemon L.) and five sweet orange (C. sinensis (L.) Osbeck) groves located in Al-Ain (south east of Abu Dhabi, UAE). Sampled trees showed weakened growth, but no typical symptoms of viroids and were tested for the presence of viroids by multiplex RT-PCR using two sets of primers specific for Citrus exocortis viroid (CEVd) and Cit- rus bent leaf viroid (CBLVd) (Al-Harthi et al., 2013). Testing disclosed the association of CEVd with 15 of 16 lemons in four groves, and with 18 of 18 sweet oranges in five groves. CBLVd was detected in six of 16 lemons in three groves and in five of 18 sweet oranges in three groves. To sequence PCR products, simplex RT-PCR was carried out with each primer set and two positive samples for CEVd and two for CBLVd. Amplifications produced fragments of the expected size, 371 bp for CEVd and 234 bp for CBLVd. PCR products were pu- rified and sequenced by Macrogen (Korea). CEVd sequences from the UAE were identical to each other and shared 99% nucleotide similarity to CEVd isolate CEVd-XNM-XY (Gen- Bank accession No. DQ431993), while the CBLVd sequence was identical to that of CBLVd isolate 201-1-2 Uy (accession No. AF428053). Sequences of both viroids from the UAE were deposited in GenBank (accession No. pending) This is the first record of CBLVd in the UAE. Presence of citrus viroids in Al-Ain, the most important agricultural area in the UAE, necessitates a larger scale study to characterize their distribution, pathogenicity and economic impact.

Multiplex detection, distribution, and genetic diversity of Hop stunt viroid and Citrus exocortis viroid infecting citrus in Taiwan

BackgroundTwo citrus viroids, Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd), have been reported and become potential threats to the citrus industry in Taiwan. The distributions and infection rates of two viroids have not been investigated since the two diseases were presented decades ago. The genetic diversities and evolutionary relationships of two viroids also remain unclear in the mix citrus planted region.MethodsMultiplex RT-PCR was used to detect the two viroids for the first time in seven main cultivars of citrus. Multiplex real-time RT-PCR quantified the distributions of two viroids in four citrus tissues. Sequence alignment and phylogenetic analysis were performed using the ClustalW and MEGA6 (neighbor-joining with p-distance model), respectively.ResultsHSVd was found more prevalent than CEVd (32.2% vs. 30.4%). Both CEVd and HSVd were commonly found simultaneously in the different citrus cultivars (up to 55%). Results of the multiplex quantitative analysis suggested that uneven distributions of both viroids with twig bark as the most appropriate material for studies involving viroid sampling such as quarantine inspection.Sequence alignment against Taiwanese isolates, along with analysis of secondary structure, revealed the existence of 10 and 5 major mutation sites in CEVd and HSVd, respectively. The mutation sites in CEVd were located at both ends of terminal and variability domains, whereas those in HSVd were situated in left terminal and pathogenicity domains. A phylogenetic analysis incorporating worldwide viroid isolates indicated three and two clusters for the Taiwanese isolates of CEVd and HSVd, respectively.ConclusionsModerately high infection and co-infection rates of two viroids in certain citrus cultivars suggest that different citrus cultivars may play important roles in viroid infection and evolution. These data also demonstrate that two multiplex molecular detection methods developed in the present study provide powerful tools to understand the genetic diversities among viroid isolates and quantify viroids in citrus host. Our field survey can help clarify citrus-viroid relationships as well as develop proper prevention strategies.

Detection of Citrus exocortis viroid on Solanum jasminoides plantlets from an Austrian nursery

Austrian Agency for Health and Food Safety (AGES), Institute of Plant Health, Spargelfeldstrasse. 191, A-1220 Vienna, AustriaInSeptember2009,thirtyplantletsofSolanumjasminoideswerereceivedfrom a commercial nursery in Upper Austria. The material originatedfrom a breeding company in Germany for growing-on and propagatingbefore reselling. The plantlets did not show any symptoms of a diseaseand were routinely tested for Potato spindle tuber viroid (PSTVd). Leafsamples were randomly taken from several plantlets and total RNA wasextractedwithacommercialRNAextractionkit(RNeasyPlantMiniKit,Qiagen),accordingtothemanufacturer’sinstructions.Aone-stepreversetranscription (RT)-PCR (Qiagen) was performed for the pospiviroidgen-eric detection using Pospi1 primers (Verhoeven et al., 2004). In parallel aRT-PCRwascarriedoutforthedetectionofPSTVdwiththeprimerspub-lished by Shamoul etal.(1997). The generic pospiviroid RT-PCR yieldedanampliconofexpectedsize.However,noPCRproductwasobtainedforPSTVd.Sequencing of the Pospi1 amplicon (partial sequence) showed a 97%identity with those of two Citrusexocortis viroid(CEVd) isolates from S.jasminoides (GenBank Accession Nos. AM920649 and AM774357).Total RNA was further tested with specific primers to CEVd (O¨ nelge,1997) and the full genome length (374 bp) was sequenced (GU300810),which exhibited 98% identity with those CEVd isolates previously men-tioned. The mismatches were on four positions: an insertion at base 49and substitutions at bases 72, 73 and 131 compared to AM920649sequence; and substitutions at the positions 73, 131, 316 and 318 com-pared to the AM774357 sequence. This is the first report of CEVd on S.jasminoides in Austria, although this pathogen had been alreadydescribedoccurringinS.jasminoidesinotherEuropeancountries(Verho-evenetal.,2008).Resultssuggest thepotentialriskofsymptomlessinfec-tion of ornamental Solanaceae by CEVd as a source of infection for cropplants,andtheurgencyofmeasurestobetakentocontroldiseasespread.AcknowledgementsWe would like to thank Mr. Ko Verhoeven (PPS, NL) for the valuablecommentsandhelpinthesequenceinterpretation.References

Suppression of Phytophthora Infection in Citrus Infected with Viroids

Citrus viroid-induced resistance to Phytophthora infection in citrus was measured by the number of Phytophthora sporangia in ‘Rio Red’ grapefruit (Citrus paradisi Macf.) bait tissue infected with citrus viroids compared with non-inoculated controls. Different viroid isolates containing mixtures of viroids [Citrus exocortis viroid (CEVd), Hop stunt viroid (HSVd), Citrus viroid III (CVd-III), Citrus viroid IV (CVd-IV)] were designated by plant numbers and sources. Source 13E was associated with the lowest number of sporangia in bark, leaves, and roots used as baits, whereas CEVd E9, a known severe CEVd isolate, significantly reduced the number of sporangia in leaves and bark. Sources 1A, 2E, 3E, 4D, and 6E showed a significantly reduced number of sporangia on bark, leaves, and roots compared with healthy plants and 44A; however, their effect was not as pronounced as that of E9 and 13E. Sources 12E and 44A did not suppress sporangia production. Previous reverse transcriptase–polymerase chain reaction analysis showed that all source plants had mixed infections with several viroids, whereas 12E and 44A contained no viroids. In addition to confirming the earlier reports on the suppression of Phytophthora infection in general, our study showed significantly reduced Phytophthora sporangia development resulting from a number of viroids in mixed infection, but there did not appear to be any effect related to viroid species. To determine if concentration affected resistance to Phytophthora, phenolic acids were extracted. Extraction of phenolic acids with 80% ethanol was more efficient compared with 100% methanol and an acetonitrile–water mixture. High-performance liquid chromatography revealed no notable detection of salicylic acid in healthy and viroid-infected plants, but there was a small peak corresponding to salicylic acid in Phytophthora-infected and both viroid and Phytophthora-infected plants. Flavone was detected in all the source plants with a slight increase in Phytophthora-infected and both viroid and Phytophthora-infected plants. A peak corresponding to quercetin dehydrate was detected in Phytophthora-infected plants. Efficient use of the right viroid isolate(s) can result in suppression of Phytophthora infection of citrus.

First Report of Solanum jasminoides Infected by Citrus exocortis viroid in Germany and the Netherlands and Tomato apical stunt viroid in Belgium and Germany.

Recent identifications of Chrysanthemum stunt viroid (CSVd) and Potato spindle tuber viroid (PSTVd) in Solanum jasminoides (3,4) prompted the testing of this plant species for infections with other pospiviroids. From autumn of 2006 to spring of 2007, samples from symptomless plants of S. jasminoides were collected in Belgium (3 samples ranging from 75 to 150 plants), Germany (3 samples ranging from 1 to 200 plants), and the Netherlands (3 samples ranging from 2 to 200 plants). Samples were tested for pospiviroids by reverse transcription (RT)-PCR assays using the Pospi1-FW/RE and Vid-FW/RE (2) and PSTV-Nb-FW (5'-ggatccccggggaaacctgga-3')/RE (5'-ggatccctgaagcgctcctcc-3') primer sets. Each set amplifies several but not all pospiviroids. The first and last primer sets amplified PCR products from six samples. The full-length genomes of all six isolates were amplified using primer pairs CEVd-FW1/RE1 (1) and CEVd-FW2 (5'-gtgctcacctgaccctgcagg-3')/RE2 (5'-accacaggaacctcaagaaag-3'), which are fully complementary to both Citrus exocortis viroid (CEVd) and Tomato apical stunt viroid (TASVd). Sequence analysis of the PCR products identified CEVd from two samples each from Germany and the Netherlands and TASVd from one sample each from Germany and Belgium (plants were imported from Israel). Although the sequences of the different CEVd isolates from S. jasminoides were not identical, all exhibited more than 95% identity with a CEVd isolate from Vicia faba (GenBank Accession No. EF494687). Both TASVd sequences were identical and showed 99.2% identity to a TASVd isolate from tomato (GenBank Accession No. AY 062121). Two nucleotide sequences of CEVd were submitted to the NCBI GenBank (Accession Nos. EU094207 and EU094208). The two other CEVd sequences and the TASVd sequence were submitted to the EMBL Nucleotide Sequence Database as Accession Nos. AM774356, AM774357, and AM777161. In addition to identification from S. jasminoides by sequence analysis, TASVd infection in the S. jasminoides sample from Germany and CEVd in one sample from the Netherlands was confirmed by mechanical inoculation to tomato followed by RT-PCR using the two CEVd-FW/RE primer pairs and analysis of the sequenced PCR product. Infection by CEVd and TASVd was also confirmed in the German samples by Northern hybridization and TASVd was confirmed in the Belgian sample by return-polyacrylamide gel electrophoresis. To our knowledge, these are the first reports of CEVd and TASVd in S. jasminoides. The viroids do not reduce the quality of S. jasminoides plants; however, the infected plants may act as infection sources for other crops.

Host Effect on the Molecular and Biological Properties of a Citrus exocortis viroid Isolate from Vicia faba

ABSTRACT Citrus exocortis viroid (CEVd) is the casual agent of citrus exocortis disease, and has been found in naturally infected citrus and noncitrus hosts. Field isolates of CEVd may infect susceptible hosts as a complex of genetically related sequence variants (haplotypes). In the present work, a CEVd isolate recovered from a symptomless broad bean plant was characterized as a heterogeneous population with a nucleotide diversity of 0.026, which did not contain a predominant haplotype. When nucleic acid extracts of this infected broad bean were used to inoculate tomato, the plants displayed symptoms and the CEVd population was more homogeneous, with a nucleotide diversity of 0.007. However, when nucleic acid extracts from this tomato isolate were back inoculated to new broad bean plants, this isolate did not revert to the original population, because it showed low nucleotide diversity (0.001) and induced symptoms in the broad bean plants. Symptomless broad bean plants may act as reservoirs of highly heterogeneous populations of CEVd variants, providing an excellent inoculum source in terms of its potential to infect a broad range of putative hosts. The epidemiological implications are discussed.

Molecular characterization of CEVd strains that induce different phenotypes in Gynura aurantiaca: structure-pathogenicity relationships

Two Citrus exocortis viroid isolates (CEVd-s and CEVd-129) that induce severe and mild symptoms in Gynura aurantiaca, respectively, have been characterized. They present nucleotide sequences in the pathogenicity motifs P(L), C and P(R) similar to those of "Class A" and "Class B". Infectivity and symptom expression in G. aurantica and tomato were evaluated with a selection of sequence variants recovered from both isolates. As expected, the two variants selected from CEVd-s induced severe symptoms. The variants selected from CEVd-129 induced mild symptoms, except one of them, named MJ, that presented an unusual genotype and induced severe symptoms in G. aurantiaca. The biological properties of MJ show that the two nucleotide changes of the C domain normally associated with the P(L) and P(R) motifs of "Class B" strains are not implicated in symptom expression. The relationship between "Class A" and "Class B" strains with the symptoms induced in clementine trees grafted on trifoliate orange is discussed.

Citrus exocortis viroid and Hop Stunt viroid Doubly infecting grapevines in Brazil

Viroids, non-protein-coding small (246-401 nt) circular single-stranded RNAs with autonomous replication, are currently classified into two families. Within the family Pospiviroidae, Citrus exocortis viroid (CEVd) belongs to the genus Pospiviroid while Hop stunt viroid (HSVd) is the single member of the genus Hostuviroid. These pathogens are distributed worldwide and infect a large number of hosts. In Brazil, isolates of CEVd and HSVd have been detected in both citrus and grapevine. To characterize and study the genetic variability of these viroids, total RNA from leaves of grapevine Vitis vinifera 'Cabernet Sauvignon' and V. labrusca 'Niagara Rosada' from Bento Gonçalves, RS, was used as a template for RT-PCR amplification with specific primers for the five viroids described infecting grapevines [HSVd, CEVd, Grapevine yellow speckle viroid 1 (GYSVd-1), Grapevine yellow speckle viroid 2 (GYSVd-2) and Australian grapevine viroid (AGVd)]. Leaf samples of Citrus medica infected with CEVd from São Paulo were also analyzed. The resulting products were separated by agarose gel electrophoresis and DNA fragments of the expected size were eluted, cloned and sequenced. The grapevine samples analyzed were doubly infected by CEVd and HSVd. A phylogenetic analysis showed that the Brazilian grapevine HSVd variants clustered with other grapevine HSVd variants, forming a specific group separated from citrus variants, whereas the Brazilian CEVd variants clustered with other citrus and grapevine variants.

Genetic variation and population structure of an isolate of Citrus exocortis viroid (CEVd) and of the progenies of two infectious sequence variants

The population structure and diversity within a Citrus exocortis viroid (CEVd) isolate was estimated by single strand conformation polymorphism (SSCP) and sequence analysis. A predominant sequence variant (V1) representing 52.8% of the overall population was identified. V1 and other additional variants presented a composition of the P domain characteristic of severe strains of CEVd. The nucleotide diversity of this CEVd population was lower than expected according to a model of neutral evolution, suggesting a strong negative selection. Citron plants were inoculated with dimeric clones of nine sequence variants and two resulted infectious inducing the severe symptoms characteristic of the original isolate. De novo populations were generated from these infectious variants and like in the original CEVd isolate, both populations presented V1 as the predominant variant but they evolved to a higher nucleotide diversity.

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.

Restoration of secondary hairpin II is associated with restoration of infectivity of a non-viable recombinant viroid

Mutagenesis and/or construction of recombinants by exchange of genomic regions between parental molecules constitute powerful tools for the study of viroids. However, a large proportion of such modifications results in molecules, which have lost their infectivity. Such is the case for a recombinant viroid named CECS, obtained by replacing the right half of a citrus exocortis viroid (CEVd) by the same region from chrysanthemum stunt viroid (CSVd). In an effort to recover viable infectious progeny from this recombinant, tomato plants were inoculated with an Agrobacterium strain carrying a dimer of the CECS viroid in positive orientation under the control of the CaMV 35S promoter. About 20% of the plants treated in this way were found to be infected with a replicating viroid, which was further propagated. Sequence analysis of six cloned full-length cDNAs derived from progeny molecules revealed the presence of mutations as compared with the parental CECS sequence. However, only two types of mutations were consistently recovered in all progeny molecules, the addition of a G in a string of four at positions 70–73, a mutation frequently observed in CEVd isolates and mutations leading to the restoration of the correct base pairing in secondary hairpin II. These results show that agro-infection is a suitable technique for the recovery of viable molecules from non-infectious viroid mutants and confirm that the ability to form secondary hairpin II is a prerequisite for viroid infectivity.

Variability of Citrus Exocortis Viroid (CEVd)

Single-strand conformation polymorphism (SSCP) has been adopted as a tool for viroid characterization. Nucleic acid extracts from a viroid field isolate containing CEVd was sub- jected to retrotranscription and PCR amplification using a set of contiguous CEVd-specific prim- ers. The analysis of cloned DNAs recovered from this isolate illustrates the extent of variability within a single CEVd isolate. The relationship between the SSCP profiles observed among clones and the variation in their nucleotide sequences was confirmed by sequence analysis of selected clones. The results indicate that field isolates are complex populations or quasispecies. Two highly conserved sequences (master sequences) as well as several less abundant variants were consis- tently identified. The sequence variants presented a base pairing arrangement in the P domain characteristic of severe strains of CEVd. A branched secondary structure rather than the rod-like structure was predicted to occur at 37 ° C. Index words. Variants, SSCP, quasispecies. Citrus exocortis viroid (CEVd), the causal agent of the exocortis dis- ease, was discovered in 1972 (12). Determination of its primary struc- ture (4, 14), showed that the CEVd molecule is a covalently closed circu- lar RNA with a highly base paired rod-like structure which conforms the model of five structural domains (T L , P, C, V, T R ) (7). Viroid replication which occurs in the absence of proof-reading mecha- nisms accounts for a high rate of variability generation. Although there are numerous structural con- straints, which impair infectivity

Direct Nucleotide Sequencing of Citrus Exocortis Viroid (CEV)

Citrus exocortis viroid (CEV), the causal agent of bark shelling disease in Poncirus trifoliata (L.) Raf, can be distinguished by its biological properties in Etrog citron and Gynura aurantiaca D. C. CEV isolate obtained from citrus trees of Çukurova region was reverse transcription-polymerase chain reaction assay (RT-PCR). The amplified products were gel purified, and subsequently sequenced without cloning, using the Promega fmol sequencing system. Both CEVc (citron) and CEVg (Gynura) were determined 371 nucleotides (nts) in length. Comparing the nucleotide sequence of CEV isolate from Çukurova region and California isolates of CEV, a number of exchanges were found in the pathogenic and variable domains.

Induced Tolerance to Mal Secco Disease in Etrog Citron and Rangpur Lime by Infection with the Citrus Exocortis Viroid

Etrog citron and Rangpur lime plants were each inoculated with one of four isolates of the citrus exocortis viroid (CEVd) and incubated for 1 yr, during which time the viroid spread systemically. CEVd caused severe leaf curling and short internodes in Etrog citron but not in Rangpur lime. After leaf inoculation with Phoma tracheiphila, typical mal secco disease symptoms developed on leaves of both cultivars, regardless of prior infection by CEVd, but growth of the mycelium from the infected leaves into the branches was greatly affected by CEVd infection. In Etrog citron, P. tracheiphila was isolated from only 9.1% of the branches sampled from CEVd-infected plants, compared with 100% from CEVd-free plants. In Rangpur lime, the incidence of branches containing P. tracheiphila varied in CEVd-infected plants from 16.7 to 50% among CEVd isolates, compared with 70% in the viroid-free controls. This is believed to be the first report of systemic resistance induced in a woody plant by prior inoculation with a viroid

Genomic Diversity Among Populations of Two Citrus Viroids from Different Graft-Transmissible Dwarfing Complexes in Israel

The nucleotide sequences of citrus bent leaf viroid (CBLVd) (formerly designated CV-Ib) and citrus exocortis viroid (CEVd), both found among the citrus viroid (CVd) populations from five graft-transmissible dwarfing complexes (GTDCs) originating from different source plants and geographical locations in Israel, were determined. The sequence homology varied only slightly among the CBLVd sequence variants, i.e., 0-7 nucleotides (nts); originating from a single GTDC and 2-8 nts between CBLVds that were obtained from different GTDCs. The lowest level of homology between CBLVd variants obtained from citrus was 97.5%. Considerably larger variation (8-15 nucleotide changes) was observed between CBLVd variants derived from citrus and the type strain that was passed through avocado seedlings. The type strain differed from all variants in six positions, located at 38, 62, 138, 179, 264, and 268 nts. The CEVd sequence variants showed considerable heterogeneity. Five variants derived from four GTDCs differed only in 2-9 nts. Five other variants derived from two GTDCs showed 27-50 nucleotide changes compared with the first group of CEVd variants. The largest variation within a single GTDC of up to 41 nucleotide changes was observed between CEVd variants derived from GTDC-G. Using CEVd # 225 as a reference strain, most of the nucleotide changes occurred in the V, LT, and RT domains. Additional changes in the P domain were found only among CEVd isolates derived from GTDC-M and GTDC-G. The sequence homologies to CEVd # 225 ranged from 89-2 (CEVd-G 2 ) to 99-0% (CEVd-NG 1 )

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.

Construction of novel viroid chimeras containing portions of tomato apical stunt and citrus exocortis viroids

Several novel tomato apical stunt viroid (TASV) recombinants were isolated after inoculation of tomato seedlings with monomeric viroid cDNAs. Two intraspecific recombinants were constructed by exchanging the left and right sides of the closely related Ivory Coast and Indonesian strains of TASV, and a third, interspecific, recombinant was constructed by similar manipulations involving TASV and citrus exocortis viroid (CEV) cDNAs. Characterization of these TASV recombinants by RNA protection assays and nucleotide sequence analysis of polymerase chain reaction-amplified cDNAs revealed no evidence for sequence instability. The symptoms induced by replication of the CEV-TASV chimera in tomato were milder than those induced by either TASV or the TASV chimeras and resembled those induced by the CEV isolate which provided its pathogenicity domain.

The sequence of a viroid from grapevine closely related to severe isolates of citrus exocortis viroid

The primary structure of a grapevine viroid (GVs) isolated in Spain was determined. The sequence consisted of 369 nucleotide residues forming a circular molecule. GVs presented extensive homology with viroids of the potato spindle tuber viroid (PSTV) group, that was specially high in the case of citrus exocortis viroid (CEV) both with variants found in isolates inducing severe (92% with CEV-A) and mild (89% with CEV-DE26) symptoms on tomato. The secondary structure proposed for GVs showed that the changes in the sequence in relation to CEV-A generated modifications of the secondary structure particularly important in the left terminal (Tl), variable (V) and pathogenesis (P) viroid domains that have been postulated. Nevertheless it was noted in GVs a central core in the P domain that is conserved in the class A sequence variants characteristic of severe isolates, but not in the class B ones found in mild isolates of CEV. These observations indicate that GVs should be considered as a severe isolate of CEV from grapevine (CEV-g), a suggestion that correlates with the biological properties of CEV-g both in tomato and in Gynura aurantiaca. The presence of this central core in the P domain seems to characterize all the variants of CEV inducing severe symptoms in tomato.

Replication of in vitro constructed viroid mutants: location of the pathogenicity-modulating domain of citrus exocortis viroid

Sequence variants from field isolates of citrus exocortis viroid (CEV) that cause either mild or severe symptoms on tomato plants have previously been classified into two groups, A and B. These groups differ primarily in two domains, P(L) and P(R), of the proposed native structure. Infectivity studies with full-length cDNA clones of variants from each class have now directly confirmed the original correlation between Class A sequences and the severe phenotype and between Class B sequences and the mild phenotype. Direct evidence for this correlation could only be obtained by using individual sequence variants since field isolates of CEV have been shown to contain a mixture of RNA species. The construction and infectivity of chimaeric cDNA clones derived from mild and severe sequence variants of CEV has demonstrated that novel, infectious viroid molecules can be generated in vitro, and that P(L) is the pathogenicity-modulating domain. The role of the P(R) domain is not known but infectivity experiments with one chimaeric cDNA clone suggest that it may influence the efficiency of the infection or replication process of the viroid in the plant.

Eleven new sequence variants of citrus exocortis viroid and the correlation of sequence with pathogenicity.

Full-length double-stranded cDNA was prepared from purified circular RNA of two new Australian field isolates of citrus exocortis viroid (CEV) using two synthetic oligodeoxynucleotide primers. The cDNA was then cloned into the phage vector M13mp9 for sequence analysis. Sequencing of nine cDNA clones of isolate CEV-DE30 and eleven cDNA clones of isolate CEV-J indicated that both isolates consisted of a mixture of viroid species and led to the discovery of eleven new sequence variants of CEV. These new variants, together with the six reported previously, form two classes of sequence which differ by a minimum of 26 nucleotides in a total of 370 to 375 residues. These two classes correlate with two biologically distinct groups when propagated on tomato plants where one produces severe symptoms and the other gives rise to mild symptoms. Two regions of the native structure of CEV, comprising 18% of the total residues, differ between the sequence variants of mild and severe isolates. Whether or not both of these regions are essential for the variation in pathogenicity has yet to be determined.