Cadang-cadang Research Articles

Novel RNA genosensor based on highly stable gold nanoparticles decorated phosphorene nanohybrid with graphene for highly sensitive and low-cost electrochemical detection of coconut cadang-cadang viroid.

Coconut cadang-cadang viroid (CCCVd) is an infectious single-stranded RNA (ssRNA) pathogen, which leads directly to the death of a large number of coconut palm trees and heavy economic loss to coconut farmers. Herein, a novel electrochemical impedance RNA genosensor is presented based on highly stable gold nanoparticles (AuNPs) decorated phosphorene (BP) nanohybrid with graphene (Gr) for highly sensitive, low-cost, and label-free detection of CCCVd. BP-AuNPs are environmentally friendly prepared by ultrasonic-assisted liquid-phase exfoliation of black phosphorus, accompanying direct reduction of chloroauric acid. Gr/BP-AuNPs are facilely prepared by the in situ growth of AuNPs onto the BP surface and its nanohybrid with Gr to improve environmental stability of BP. Gr/BP-AuNP-based RNA genosensor is fabricated by immobilizing the thiol-functionalized single-stranded DNA (ssDNA) oligonucleotide probe onto the surface of Gr/BP-AuNP-modified glassy carbon electrode via gold-thiol interactions, which served as an electrochemical genosensing platform for the label-free impedance detection of CCCVd by hybridization between the functionalized ssDNA probe and the complementary CCCVd ssRNA sequence in a wide linear range from 1.0 × 10-11 to 1.0 × 10-7M with a low limit of detection of 2.8 × 10-12M. This work supplies an experimental support and theoretical direction for the fabrication of RNA biosensors based on graphene-like materials and potential application for a specific diagnosis of plant RNA viral disease in Arecaceae planting industry.

Analysis of Coconut cadang-cadang viroid variants on field samples exhibiting variation in orange spotting symptom expression and severity.

Sequence variation has been attributed to symptom variations but has not been investigated in Orange Spotting-Coconut cadang-cadang viroid (OS-CCCVd) infected palms. Likewise, the relationship between Coconut cadang-cadang viroid (CCCVd) variants, Orange Spotting (OS) severity and the accumulation of the viroid in the palms have not been elucidated. This paper describes the characterization of CCCVd variants by cloning and sequencing, followed by correlation with symptom expression. Total nucleic acids were extracted from leaf samples harvested from frond 20 of seven Dura×Pisifera (D×P) African oil palm (Elaeis guineensis Jacq.) aged between 13 and 21years old collected from local plantations. The nucleic acids were fractionated using 5% non-denaturing polyacrylamide gel electrophoresis (PAGE) before being subjected to detection by reverse transcribed polymerase chain reaction (RT-PCR). The PCR products were cloned into a plasmid vector and the sequence of the clones was analyzed. CCCVd variants were quantified using real-time qPCR assay with CCCVd specific primers. Sixteen randomly selected clones of (OP246) had an arbitrary 100% identity with CCCVdOP246 (GeneBank Accession No: HQ608513). Meanwhile, four clones had >93% similarity with several minor sequence variations forming variants of OP234, OP235, OP251 and OP279. The OS symptoms observed in the field were characterized into three categories based on the size and morphology of the orange spots on the affected fronds. In addition, there was no direct correlation between disease severity and the accumulation of CCCVd variants in oil palm. This finding is the first report describing the sequence variation of the CCCVd RNA and symptom variation in OS oil palm field samples.

Pest categorisation of coconut cadang-cadang viroid.

The EFSA Panel on Plant Health updated its 2017 pest categorisation of coconut cadang cadang viroid (CCCVd) for the EU territory due to new data on its host range. The identity of CCCVd, a member of the genus Cocadviroid (family Pospiviroidae), is established and detection and identification methods are available. It is included as a quarantine pest for the EU in the Commission Implementing Regulation (EU) 2019/2072. CCCVd has been reported from the Philippines and Malaysia. It is not known to be present in the EU. The host range of CCCVd is restricted to Arecaceae species (palms), in particular, coconut palm (Cocos nucifera) to which it causes a lethal disease. Oil palm (Elaeis guineensis) and buri palm (Corypha utan) are other natural hosts of CCCVd. Palm species of several genera, including Phoenix spp. and other species grown and/or cultivated in the EU, have been identified as potential hosts. The viroid is naturally transmitted at low rate by seeds and pollen and possibly by additional not yet identified natural transmission means. It can be transmitted through vegetative propagation applied to some palm species. Plants for planting including seeds of its hosts have been identified as the main entry pathway of CCCVd. Potential hosts of CCCVd are present in the EU, therefore establishment is possible. Should the pest establish in the EU, an impact is expected, with uncertainty on its magnitude. The Panel identified the susceptibility of palm species grown in the EU as a key uncertainty potentially affecting the conclusion of this pest categorisation. Nevertheless, the pest satisfies the criteria that are within the remit of EFSA to assess for this viroid to be regarded as potential Union quarantine pest.

Estimating chlorophyll content at leaf scale in viroid-inoculated oil palm seedlings (Elaeis guineensis Jacq.) using reflectance spectra (400 nm–1050 nm)

ABSTRACTOrange Spotting (OS) is an emerging disease in oil palm (Elaeis guineensis Jacq) that has been associated with Coconut cadang-cadang viroid (CCCVd; Cocadviroid, Pospiviroidae). This study was aimed at selecting efficient wavebands in the Visible/near infrared (VNIR) spectrum (400–1050 nm) for estimation of chlorophyll content in CCCVd-inoculated oil palm seedlings. The VNIR spectrum is most sensitive to chlorophyll stress and able to differentiate between healthy and diseased plants. Therefore, a greenhouse experiment was conducted to remotely sense the chlorophyll content using a hyperspectral hand-held spectroradiometer (Analytical Spectral Device (ASD) FieldSpec–II) and a portable chlorophyll meter (Minolta Soil-Plant Analysis Development (SPAD)−502). FieldSpec–II provides reflectance spectra with narrow and contiguous wavebands while SPAD-502 churns out SPAD readings corresponding to chlorophyll content. Interval Partial Least Squares (iPLS) model was used to assess the relationship between independent variable (spectra) and dependent variable (SPAD readings). Five different spectral datasets (i.e. raw spectra, first-order derivatives, Savitzky-Golay smoothing, Multiplicative Scatter Correction (MSC), and standard normal transformation) were investigated. Results showed that the MSC dataset yielded the most accurate estimation with a root mean square error of prediction of 3.70% and a correlation coefficient for prediction of 0.72. A total of thirty wavebands within a range between 601 nm and 630 nm was selected. This work showed that VNIR spectra has the potential to diagnose OS disease in oil palm using the iPLS regression method. This is completely a new application of hyperspectral remote sensing for the oil palm industry, particularly in the domain of plant protection.

Selection of a Spectral Index for Detection of Orange Spotting Disease in Oil Palm (Elaeis guineensis Jacq.) Using Red Edge and Neural Network Techniques

Spectral screening can play an important role in successful detection of viroid-infected oil palm seedlings from nursery stage prior to transplanting into the field. Coconut cadang–cadang viroid (CCCVd) is the main causal agent of orange spotting (OS) disease. OS disease is an emerging disease in Malaysian plantation. In this study, a glasshouse experiment was conducted with fifteen CCCVd-inoculated and five healthy oil palm seedlings in the growing season of 2015. Spectral screening was performed using a hyperspectral spectroradiometer, Analytic Spectral Device HandHeld 2 (325–1075 nm). The red edge, a steep gradient in reflectance between red and near-infrared bands (680–780 nm), was used for selection of red edge bands. A maximum point (i.e., 700 nm) and minimum point (i.e., 768 nm) of red edge were selected from healthy and inoculated spectra. Shifts of red edge inflection point from healthy to inoculated spectra were also studied. Four well-known spectral indices, namely simple ratio, red edge normalized difference vegetation index, two-band enhanced vegetation index 2 (EVI2), and chlorophyll index red edge, were evaluated using selected red edge bands. The multilayer perceptron neural network model was used to establish a nonlinear relationship between selected spectral bands and each spectral index. EVI2 was selected as a best spectral index which resulted in zero errors at the training, testing, and validation datasets. The highest coefficient of correlation (r = 1) was recorded between spectral bands (input values) and EVI2 (target values).

Pest categorisation of Cadang-Cadang viroid.

The EFSA Panel on Plant Health performed a pest categorisation of Cadang‐Cadang viroid for the European Union (EU) territory. Coconut cadang‐cadang viroid (CCCVd) is a well‐known viroid for which efficient molecular detection assays are available. It is transmitted by vegetative multiplication of infected hosts, by seed and pollen and, possibly, by the action of unknown vector(s). CCCVd is reported from a few countries in Asia and is not known to occur in the EU. It therefore does not meet one of the criteria for being a Union regulated non‐quarantine pest. The host range of CCCVd is restricted to Arecaceae species (palms), in particular coconut and it is listed on all known hosts in Annex IIAI of Directive 2000/29/EC. CCCVd is expected to be able to enter in the EU and to be able to establish in the open in the southernmost regions of the EU and elsewhere under protected cultivation. It has the potential to subsequently spread via plants for planting and possibly other mechanisms. CCCVd is able to cause severe symptoms in some Arecaceae species while others seem less affected. The potential impact of CCCVd if introduced in the EU is very difficult to assess. Given that the spread potential is, as for other viroids, likely to be limited, the potential impact is estimated to be limited in extent but this judgement is affected by large uncertainties. Overall, CCCVd meets all the criteria evaluated by EFSA to qualify as Union quarantine pest. The main knowledge gaps concern (1) the relationships between CCCVd‐related RNAs and CCCVd, (2) the origin and volume of the trade in palm seeds and plants for planting imported in the EU (3) the efficiency of natural spread under EU conditions and (4) host status and susceptibility of many palm species grown in the EU.

Optimization of total nucleic acid extraction method for detecting Coconut cadang-cadang viroid variants in oil palm

Variants of Coconut cadang-cadang viroid (CCCVd) with over 90% sequence similarity to CCCVd in coconut have recently been associated with orange spotting disease in oil palm, especially in low concentrations in infected hosts. Thus, there is a need to extract high-quality nucleic acid for molecular detection of the viroid. Total nucleic acid (TNA) was extracted from oil palm leaf samples with orange spotting symptoms collected from different states in Malaysia using a modified and optimized version of the conventional natrium chloride EDTA Tris-HCL mercaptoethanol extraction method. The modifications involved additional ethanol and lithium chloride precipitation stages, thereby eliminating the need for further purification through non-denaturing polyacrylamide gel electrophoresis (PAGE). The modified procedure yielded a mean volume of 60 μg RNA per 10 g of fresh tissue which is approximately four times the volume produced by the conventional extraction method. Furthermore, the modified method resulted in higher purity than the conventional method according to the ratios of absorbance at wavelengths of 260/280 nm and 260/230 nm, which were 1.838 and 1.883 with the modified method and 1.085 and 0.765 with the conventional method. The modified method enabled extraction of high-quality RNA from all samples investigated. The extracted RNA was suitable for cDNA synthesis and PCR amplification and showed consistent detection of CCCVd-like RNA at approximately 250 amplicons. Using the conventional method, detection of CCCVd-like RNA was inconsistent and only feasible after the PAGE purification step.

Sensitivity enhancement of graphene/zinc oxide nanocomposite-based electrochemical impedance genosensor for single stranded RNA detection

An efficient electrochemical impedance genosensing platform has been constructed based on graphene/zinc oxide nanocomposite produced via a facile and green approach. Highly pristine graphene was synthesised from graphite through liquid phase sonication and then mixed with zinc acetate hexahydrate for the synthesis of graphene/zinc oxide nanocomposite by solvothermal growth. The as-synthesised graphene/zinc oxide nanocomposite was characterised with scanning electron microscopy (SEM), transmission electron microscopy (TEM), Raman spectroscopy and X-ray diffractometry (XRD) to evaluate its morphology, crystallinity, composition and purity. An amino-modified single stranded DNA oligonucleotide probe synthesised based on complementary Coconut Cadang-Cadang Viroid (CCCVd) RNA sequence, was covalently bonded onto the surface of graphene/zinc oxide nanocomposite by the bio-linker 1-pyrenebutyric acid N-hydroxysuccinimide ester. The hybridisation events were monitored by electrochemical impedance spectroscopy (EIS). Under optimised sensing conditions, the single stranded CCCVd RNA oligonucleotide target could be quantified in a wide range of 1.0×10–11M to 1.0×10−6 with good linearity (R =0.9927), high sensitivity with low detection limit of 4.3×10–12M. Differential pulse voltammetry (DPV) was also performed for the estimation of nucleic acid density on the graphene/zinc oxide nanocomposite-modified sensing platform. The current work demonstrates an important advancement towards the development of a sensitive detection assay for various diseases involving RNA agents such as CCCVd in the future.

Use of Reflectance Spectroscopy as a Tool for Screening Viroid-Inoculated Oil Palm Seedlings

Reflectance spectroscopy was assessed as a new diagnostic tool for screening oil palm (Elaeis guineensis Jacq.) seedlings inoculated with Coconut cadang-cadang viroid (CCCVd). This study was aimed at non-destructive diagnosis of Orange Spotting (OS) disease of oil palm under glasshouse conditions. Leaf reflectance (in the detection range of 325-1075 nm) was acquired from oil palm seedlings with and without inoculation (control) of CCCVd. Spectral reflectance was measured at 15 and 30 days after inoculation using a non-imaging spectroradiometer, ASD FieldSpec® HandHeld™ 2. The red-edge region (680-780 nm) was investigated, and two spectral bands (i.e. 680 nm and 754 nm) were selected. Reflectance Sensitivity (RS) analysis was performed over these spectral bands. The far red band at 680 nm was found to be markedly sensitive to CCCVd infection while the spectral band at 754 nm was found to be insensitive. The 680 nm band gave an RS of 35.4% while the 754 nm band registered an RS of -5%. This study demonstrates the potential of screening CCCVd infection at an early stage (nursery establishment) non-destructively.

Comparison of RNA extraction methods for RT-PCR detection of Coconut cadang-cadang viroid variant in orange spotting oil palm leaves

Coconut cadang-cadang viroid (CCCVd) is a lethal disease that has devastated the coconut industry in the Philippines. Recently, a CCCVd variant associated with orange spotting of foliage, which is a phenomenon reported in the Malaysian oil palm industry, occurring as an isolated palm with no similar symptoms on the adjacent neighbouring palms. The presence of this viroid in oil palms is difficult to detect as it is found in very low concentrations. Thus, an efficient RNA extraction method for isolating the CCCVd variant and to detect it through reverse transcriptase polymerase chain reaction (RT-PCR) using specific primers for CCCVd was required. Leaf samples from symptomatic and asymptomatic oil palms were collected from various locations in Malaysia, namely, Negeri Sembilan, Selangor and Perak. The samples were subjected to three RNA extraction methods – natrium chloride EDTA Tris-HCl mercaptoethanol extraction (NETME), polyethylene glycol extraction (PEG) and cetyltrimethylammonium bromide extraction (CTAB). The quantification of the RNA, based on optical density (OD) and concentration, showed that the CTAB extraction was the best, followed by the NETME extraction. The RNA extraction using PEG resulted in poor yields and had the lowest purity. The viroid was detected in all of the samples extracted via CTAB and NETME by RT-PCR with reduced starting material compared with PEG extracted RNA. A BLAST analysis indicated that the viroid sequences are highly conserved and shared a 93% sequence identity with the CCCVd246 variant from oil palm. Phylogenetic analysis showed that the variant belonged to an outgroup from other cocadviroids found in monocots.

The discovery and eradication of potato spindle tuber viroid in Canada.

In 1960s, potato spindle tuber was thought to be a viral disease. In 1971, the agent of the disease was characterised as a low-molecular weight infectious ribonucleic acid (RNA), which was named as 'viroid', specifically Potato spindle tuber viroid (PSTVd)). Since then, more than 30 plant diseases in horticultural and ornamental plants have been shown to be caused by different viroids globally. Viroids are single-stranded RNA, covalently closed circular molecule, without any protein coat. They are the smallest known plant pathogen containing RNA genome ranging from 246 nucleotides (Coconut cadang-cadang viroid) to 399 nucleotides (Chrysanthemum chlorotic mottle viroid). Some viroids are located in the plant cell nucleus (pospiviroids) and others in the chloroplast (avsunviroids). With the recognition of pathogenic nature of viroid, specific detection methodologies were developed, which enabled detection of PSTVd in seed-potato tubers prior to their planting in the field, and thus PSTVd was prevented from spreading the disease. As a result, PSTVd was eradicated from Canada in late 1980s. Viroids similar to PSTVd (Pospiviroid) have been discovered and they are detected in symptomless ornamental plants. Although, PSTVd has been eradicated from Canada, there is a strong possibility of viroid introduction from other plants besides potato and tomato and causing PSTVd like diseases.

Detection of Coconut cadang-cadang viroid (CCCVd) in oil palm by reverse transcription loop-mediated isothermal amplification (RT-LAMP)

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) detected Coconut cadang-cadang viroid (CCCVd) within 60min at 60°C in total nucleic acid extracted from oil palm leaves infected with CCCVd. Positive reactions showed colour change from orange to green in the reaction mix after the addition of fluorescent reagent, and a laddering pattern band on 2% agarose gel electrophoresis. Conventional RT-PCR with LAMP primers produced amplicons with a sequence identical to the 297-nt CCCVd oil palm variant with the primers being specific for CCCVd and not for other viroids such as PSTVd and CEVd. RT-LAMP was found to be rapid and specific for detecting oil palm CCCVd.

Characterization of Coconut cadang-cadang viroid variants from oil palm affected by orange spotting disease in Malaysia

A 246-nt variant of Coconut cadang-cadang viroid (CCCVd) has been identified and described from oil palms with orange spotting symptoms in Malaysia. Compared with the 246-nt form of CCCVd from coconut, the oil palm variant substituted C(31)→U in the pathogenicity domain and G(70)→C in the central conserved domain. This is the first sequence reported for a 246-nt variant of CCCVd in oil palms expressing orange spotting symptoms.

Detection of Coconut cadang‐cadang viroid sequences in oil and coconut palm by ribonuclease protection assay

AbstractA ribonuclease protection assay (RPA) has been developed for detecting Coconut cadang‐cadang viroid (CCCVd) sequences. An RNA probe complementary to full‐length CCCVd246 was used, terminating at nucleotide 65 in the upper conserved region, and linked to a non‐viroid 5′ sequence, which acted as an internal control for ribonuclease activity. Extracts from CCCVd‐infected coconut (Cocos nucifera) and African oil (Elaeis guineensis) palms protected three major fragments of approximately 250, 125 and 50 nt and a variable number of minor fragments. Extracts of healthy coconut palms, Potato spindle tuber viroid‐infected tomato and transfer RNA did not protect the probe. The approximately 250 nt fragment is predicted to indicate the presence of monomers and dimers of circular CCCVd246, linear CCCVd246 with the same termini as the probe and point mutants of these forms. The origin of smaller protected fragments is discussed. RPA‐detected CCCVd sequences in 13 of 18 oil palms surveyed in a commercial plantation in Malaysia. Signal intensity varied between the positive oil palms and was generally lower than in coconut palms infected with CCCVd. An infection phenotype was implied but not confirmed by the observation that in a group of 10 oil palms with orange leaf spotting, 9 contained CCCVd, whereas in a group of 8 palms without orange spotting, the viroid was detected in 4. Of four coconut palms in Sri Lanka shown by dot‐blot assay to contain CCCVd‐related RNA, one was shown by RPA to be positive for the CCCVd246 sequence. RPA is therefore a robust and sensitive test for CCCVd sequences, and our results show that sequences closely related to CCCVd246 are not confined to the Philippines.

Variants of Coconut cadang-cadang viroid isolated from an African oil palm (Elaies guineensis Jacq.) in Malaysia

Variants of Coconut cadang-cadang viroid have been identified in a plantation oil palm growing in Malaysia. Three size classes are described, comprising 297, 293, and 270 nt. Compared with the 296-nt form of coconut cadang-cadang viroid (CCCVd), all variants substituted C31 --> U in the pathogenicity domain and A175 --> C in the right-hand terminus. Other mutations and deletions accounted for the different sizes. These are the first sequences reported for variants of Coconut cadang-cadang viroid in a species other than coconut palm, and this is the first evidence that variants closely related to CCCVd occur outside the Philippines.

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.

Specific Identification of Coconut Tinangaja Viroid for Differential Field Diagnosis of Viroids in Coconut Palm

ABSTRACT Tinangaja is a widespread lethal disease of putative viroid etiology affecting coconut palm on the island of Guam. Determination of its distribution and mode of spread requires a simple and reliable diagnostic procedure that is specific for the associated coconut tinangaja viroid (CTiVd). A method of extracting tissue followed by analytical agarose gel electrophoresis for CTiVd detection has been developed and used to identify the viroid in leaf samples of suspect symptomatic palms growing in the field. Two-dimensional polyacrylamide gel electrophoresis showed that the viroid band contained circular molecules that are typical for viroids. Confirmation of the identity of CTiVd and detection of low levels of viroid below the threshold of detection by agarose gel electrophoresis was achieved either by diagnostic oligonucleotide-probe (DOP) hybridization assay or by reverse-transcription polymerase chain reaction (RT-PCR) with the oligonucleotide probe as one of the two PCR primers. RT-PCR was not substantially more sensitive than DOP-hybridization assay. This procedure also was applicable to coconut cadang-cadang viroid (CCCVd), and oligonucleotide probes designed to be specific for either CTiVd or CCCVd distinguished between these two viroids in coconut leaf extracts. This strategy provides a rapid and specific indexing procedure for the two characterized viroids of coconut palm and will be applicable to further studies on the viroid-like sequences previously reported in tropical monocotyledons.

Synthesis and two-dimensional electrophoretic analysis of mixed populations of circular and linear RNAs

Spontaneous cleavage of the less abundant form of tobacco ringspot virus satellite RNA is readily reversible. Capitalizing on earlier observations by Feldstein and Bruening that small 'mini-monomer' RNAs derived from this molecule and containing little more than covalently attached ribozyme and substrate cleavage products are able to efficiently circularize, we have constructed a series of self-circularizing RNAs of precisely known size. Mixtures of linear and circular RNAs synthesized in vitro and containing 225-1132 nt could be completely resolved using a novel two-dimensional denaturing polyacrylamide gel electrophoresis system. Similar analyses of a complex mixture of coconut cadang-cadang viroid RNAs revealed the presence of relatively large amounts of a previously undescribed 'fast-slow' heterodimeric RNA species in infected palms. Only a single DNA template is required to prepare each pair of circular and linear RNA markers.

Tissue and intra‐cellular distribution of coconut cadang cadang viroid and citrus exocortis viroid determined by in situ hybridization and confocal laser scanning and transmission electron microscopy

Confocal laser scanning microscopy and transmission electron microscopy (TEM) were used in conjunction with in situ hybridization techniques to compare and contrast the subnuclear (ultrastructural) and tissue (histological) localizations, respectively, of citrus exocortis viroid (CEV) and coconut cadang cadang viroid (CCCV). Both these viroids, which are members of the same taxonomic subgroup of viroids, were found in the vascular tissues as well as in the nuclei of mesophyll cells of infected host plants. At the subnuclear level, however, CEV was distributed across the entire nucleus, in contrast to CCCV which was mostly concentrated in the nucleolus with the remainder distributed throughout the nucleoplasm.

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.