Citrus Exocortis Research Articles

Graft-transmissible citrus diseases in P. R. China-research developments

In the P.R. China, ten graft-transmissible pathogens have been identified towards citrus, including Candidatus Liberibacter asiaticus, Citrus tristeza virus (CTV), Citrus tatter-leaf virus (CTLV), Citrus exocortis viroid (CEVd), Citrus yellow vein clearing virus (CYVCV),Satsuma dwarf virus (SDV), Citrus vein enation virus (CVED), Citrus psorosis virus (CPsV), Citrus cachexia viroid (CCaVd) and Citrus chlorotic dwarf virus (CCDV). Of these pathogens, the first five cause damage in field citrus trees, whereas the latter five were occasionally detected from the imported citrus materials or field trees. The research progresses about HLB, CTV, CYVCV etc. within recent three years have briefly been reviewed.

A survey of Florida citrus viruses and viroids

Efficient disease management is critical in the production of citrus; a crop that is susceptible to several plant pathogens. The ongoing battle with citrus greening has led to a shift in cultural practices, which could lead to a resurgence of previously controlled diseases. Here we investigated the presence of several common citrus-infecting viruses and viroids (Citrus leaf blotch virus, Apple stem grooving virus (synonym: Citrus tatter leaf virus), Citrus exocortis viroid, Hop stunt viroid (synonym: Citrus viroid II), and Citrus dwarfing viroid (synonym: Citrus viroid III) in Florida citrus groves. All five viruses and viroids are still present, with varying incidence. It would be prudent to take them into consideration when developing citrus disease management strategies.

The effect of viroid infection of citrus trees on the amoebicidal activity of ‘Maltese half-blood’ (Citrus sinensis) against trophozoite stage of Acanthamoeba castellanii Neff

In order to promote a local Tunisian product, this study was designed to examine, for the first time, the anti-Acanthamoeba activity (Acanthamoeba castellanii Neff) of the essential oils of Tunisian Citrus sinensis peels (Maltese half-blood) and the effect of viroid plant infection on this activity. To do so, three samples of peels’ essential oils were studied: from a healthy plant (Control), a plant inoculated with Citrus exocortis viroid (CEVd) and one inoculated with hot stunt cachexia viroid (HSVd). The samples were extracted by hydrodistillation from dried peels and characterized by GC–MS. Limonene was the major component with a percentage ranging from 90.76 to 93.34% for (CEVd) sample and (Control), respectively. Anti-Acanthamoeba activity of the tested oils was determined by the Alamar Blue® assay. Primary results showed a strong potential anti-Acanthamoeba activity with an IC50 ranging from 36.6 to 54.58 μg/ml for (HSVd) and (CEVd) samples, respectively.In terms of the effect of viroid infection, a strong positive correlation was observed between different chemical classes and anti-Acanthamoeba activity.

The occurrence of viruses and viroids in ornamental citrus mother plants in Tuscany (Central Italy)

Citrus tristeza closterovirus (CTV) has been found several times in the last decades in Italy, and plant protection services are involved in monitoring and surveillance. Although orchards linked to the citrus industry are well monitored, there is an underestimated risk of viruses or virus-like diseases in ornamental nurseries. Our aim was to modify a CTV monitoring program to include other viruses (Citrus variegation virus, CVV; Citrus psorosis virus, CPsV) and viroids (Citrus exocortis viroid, CEVd; Hop stunt viroid, HSVd; Citrus bent leaf viroid, CBLVd; Citrus dwarfing viroid, CDVd; Citrus bark cracking viroid, CBCVd). Ornamental mother plants were monitored for four years in 15 nurseries in two locations in central Italy using inexpensive multiplex RT-PCR protocols. CTV incidence was 1.6–13.5%, with an average distribution of 11.9%. The average incidence of CVV and CPsV was 6.3% and 2.7%, respectively. Higher CTV, CVV and CPsV incidences were observed in C. x paradisi, C. grandis and C. x clementina. The most widespread viroid identified was CEVd (32.9%), frequently observed in C. x limonia and C. limon. HSVd (10.5%), and CDVd (7.1%) were mostly found in C. x limonia. Lower infection rates were observed for CBLVd (2.0%) and CBCVd (1.4%). However, the nurseries’ response to the virus alert by the protection services was only partially effective. Although the CTV incidence was lower in nurseries re-checked after the initial detection, it was not eradicated from two nurseries out of three, and the occurrence of viroids was reduced in just one nursery. Given that dangerous viruses along with the concomitant spread of viroids have unfortunately become a fact of every day life, multiplex RT-PCR diagnoses are likely to play an increasing role in warning nursery managers of possible infections.

One-step multiplex RT-qPCR detects three citrus viroids from different genera in a wide range of hosts

A one-step multiplex reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) based on species-specific minor groove binding (MGB) probes, was developed for the simultaneous detection, identification, and quantification of three citrus viroids belonging to different genera. Citrus exocortis viroid (Pospiviroid), Hop stunt viroid (Hostuviroid), and Citrus bark cracking viroid (Cocadviroid) cause a variety of maladies in agriculturally significant crops. Therefore, reliable assays for their detection are essential tools for various government and industry organizations implementing disease management programs. Singleplex qPCR primers and MGB probes were designed individually for the detection of the three targeted viroids, and subsequently combined in a one-step multiplex RT-qPCR reaction. A wide host range of woody plants, including citrus, grapevines, apricots, plums and herbaceous plants such as tomato, cucumber, eggplant and chrysanthemum different world regions were used to validate the assay. Single, double and triple viroid infections were identified in the tested samples. The developed multiplex RT-qPCR assay was compared with a previously reported SYBR Green I RT-qPCR for the universal detection of citrus viroids. Both assays accurately identified all citrus viroid infected samples. The multiplex assay complemented the SYBR Green I universal detection assay by differentiating among citrus viroid species in the positive samples. The developed multiplex RT-qPCR assay has the potential to simultaneously detect each targeted viroid and could be used in high throughput screenings for citrus viroids in field surveys, germplasm banks, nurseries and other viroid disease management programs.

Phylogeny of five predominant pospiviroid species in Belgium

In Belgium pospiviroids are routinely detected in various hosts. The most frequently found pospiviroids are: Citrus exocortis viroid (CEVd), Chrysanthemum stunt viroid (CSVd), Potato spindle tuber viroid (PSTVd), Tomato apical stunt viroid (TASVd) and Tomato chlorotic dwarf viroid (TCDVd). Apart from the high incidence of pospiviroids in latently-infected ornamentals, viroids have also been found in plants where they cause disease: PSTVd and TCDVd in tomatoes and CSVd in chrysanthemum. In order to gain more epidemiological data on these infections, this study has conducted phylogenetic analyses of Belgian isolates for each of these five pospiviroid species. PSTVd and CEVd-isolates show a clustering depending on host plant identity. This was not observed for TCDVd and TASVd. A very high degree of sequence similarity was noticeable for CSVd-isolates from various hosts. During the past decade, PSTVd and CSVd-infected mother plants have been systematically eradicated in Belgium after positive detection results, also when found in symptomless plants, leading to a decreased trend of these quarantine pests in the past few years. However, other non-quarantine pospiviroid species are still ubiquitously present in many ornamentals. Since these pospiviroids can be equally harmful to crops as the two quarantine pests PSTVd and CSVd, there is still a risk that transmission occurs from symptomless-infected ornamental plants to economically important crops in Belgium such as tomato, pepper and chrysanthemum.

Salicylic Acid Is Involved in the Basal Resistance of Tomato Plants to Citrus Exocortis Viroid and Tomato Spotted Wilt Virus.

Tomato plants expressing the NahG transgene, which prevents accumulation of endogenous salicylic acid (SA), were used to study the importance of the SA signalling pathway in basal defence against Citrus Exocortis Viroid (CEVd) or Tomato Spotted Wilt Virus (TSWV). The lack of SA accumulation in the CEVd- or TSWV-infected NahG tomato plants led to an early and dramatic disease phenotype, as compared to that observed in the corresponding parental Money Maker. Addition of acibenzolar-S-methyl, a benzothiadiazole (BTH), which activates the systemic acquired resistance pathway downstream of SA signalling, improves resistance of NahG tomato plants to CEVd and TSWV. CEVd and TSWV inoculation induced the accumulation of the hydroxycinnamic amides p-coumaroyltyramine, feruloyltyramine, caffeoylputrescine, and feruloylputrescine, and the defence related proteins PR1 and P23 in NahG plants earlier and with more intensity than in Money Maker plants, indicating that SA is not essential for the induction of these plant defence metabolites and proteins. In addition, NahG plants produced very high levels of ethylene upon CEVd or TSWV infection when compared with infected Money Maker plants, indicating that the absence of SA produced additional effects on other metabolic pathways. This is the first report to show that SA is an important component of basal resistance of tomato plants to both CEVd and TSWV, indicating that SA-dependent defence mechanisms play a key role in limiting the severity of symptoms in CEVd- and TSWV-infected NahG tomato plants.

First Report of Citrus Exocortis Viroid and Two Citrus Variants of the Hop Stunt Viroid on Lemon in Azerbaijan

First Report of Citrus Exocortis Viroid and Two Citrus Variants of the Hop Stunt Viroid on Lemon in Azerbaijan

Attempts to eradicate graft-transmissible infections through somatic embryogenesis in Citrus ssp. and analysis of genetic stability of regenerated plants

Stigma/style somatic embryogenesis (SE) is proved to be effective in the complete elimination of the main Citrus virus and virus-like diseases in 100 % of analysed cases. In the present research SE was applied on 13 genotypes, belonging to the Algerian germplasm collection of two different Citrus species (Citrus limon and C. sinensis) infected by one or more graft-transmissible agents, to evaluate the genetic stability and the sanitation of regenerants. The infected genotypes were regenerated through stigma/style SE and the obtained plants were tested by serological, molecular and biological assays, 6 and 18 months after plantlet grafting, for assessing the elimination of the virus, viroid and virus-like agents present in the mother plants. No evidence of infections of virus and virus–like agents was present in the regenerated plants; whereas, after 18 months up to 24 % of tested plants were found to be infected by viroids, independently from the species and genotypes of the mother plants. Hop stunt viroid (HSVd) proved to be the most infectious viroid; conversely, Citrus exocortis viroid (CEVd) and Citrus viroid III (CVd-III) were present only in ‘Secile’ lemon, as mixed infection with HSVd. SE from stigma/style explants was 100 % effective in the elimination of mixed viroid infections in two lemons (‘Lunario’ and ‘Sans pepins’) and one orange (‘Mitidja navel’). To evaluate genetic stability of regenerants, DNA analyses were performed. No somaclonal variability was observed in lemon regenerants. However, the amplification products of ‘Washington navel 251’ (C. sinensis) revealed genetic instability in some of the regenerated plants.

Construction and application of infectious citrus viroids for biological indexing

Author(s): Steyn, C; Cook, G; Burger, J T; Maree, H J | Abstract: Viroid species identified in citrus induce a range of symptoms in this host as well as in non-citrus hosts. Currently, 7 citrus viroid species are recognized including Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus dwarfing viroid (CDVd), Citrus bark cracking viroid (CBCVd), Citrus viroid V (CVd-V), Citrus viroid VI (CVd-VI) and Citrus exocortis viroid (CEVd). Cachexia-inducing variants of HSVd and CEVd, which causes exocortis, are considered severe pathogens of citrus, whereas other viroid species induce less severe symptoms such as stunting, either singly or in combination. Some viroid species, such as CDVd, have previously been used to deliberately induce stunting for high density planting of citrus, but studies on the effects of commercial orchards are limited. Research on the effect of viroid species requires the isolation of single viroid species. Biological isolation of single species from naturally infected citrus is challenging since viroids seldom occur as single infections and are often found in combination with various other pathogens. The production of single viroid species clones for in vitro transcription enables the generation of a single viroid species inoculum for research applications and circumvents the need for maintenance of sources in plants. Complete genomes of 7 viroid strains including CBLVd, 2 HSVd variants (CVd-IIa and CVd-IIb), CDVd, CBCVd, CVd-V and CEVd were cloned with a leading T7 promoter sequence to facilitate in vitro transcription. Circularized RNA transcripts were successfully used to transfect ‘Etrog’ citron (Citrus medica) by slash-inoculation that developed typical citrus viroid symptoms.

Detection and molecular characterization of Grapevine yellow speckle viroid 1 isolates infecting grapevines in Brazil

Presently, Hop stunt viroid (HSVd) and Citrus exocortis viroid (CEVd) are the only viroids reported to infect grapevines (Vitis spp.) in Brazil, among the seven viroid species already reported infecting this host in other countries. All grapevine viroid diseases are graft-transmissible and can induce losses especially when associated with viruses. The aim of this work was to confirm infection by Grapevine yellow speckle viroid 1 (GYSVd-1) in grapevine samples exhibiting yellow speckle symptoms in the leaves and in asymptomatic samples sequenced by next generation sequencing (NGS). The occurrence of this viroid in Brazil was further investigated in a second study. Total RNAs and dsRNAs were extracted from five symptomatic plants and 16 asymptomatic samples, respectively. Specific primers were used for RT-PCR and amplified DNA fragments were cloned and sequenced by the Sanger method. Eleven complete nucleotide sequences of GYSVd-1 isolates (366–367 nt) were obtained from NGS and from RT-PCR amplicons. Comparisons showed high identities (95.9–100 %) among ten isolates and an identity of 87.2–90.4 % with a divergent isolate (RM-BR). Phylogenetic analyses placed GYSVd-1 isolates in four clusters (types 1, 2, 3 and 4). All GYSVd-1 infections were confirmed by conventional RT-PCR and RT-qPCR using specific oligonucleotides and a labeled probe. This is the first report and molecular characterization of GYSVd-1 infecting grapevines in Brazil, and our survey indicates that this viroid could be widespread in the major grape producing regions of Brazil.

FIRST IDENTIFICATION OF CITRUS EXOCORTIS VIROID (CEVd) AND CITRUS DWARF VIROID (CVd-III) IN CITRUS ORCHARDS IN SYRIA

Following the first finding of Hop stunt viroid (HSVd) in Syria (Abou Kubaa et al., 2011), a survey of citrus was conducted during the summer 2013 to investigate the presence of other viroids in the country. Leaf samples were collected from 65 stunted trees from four commercial orchards located in Lattakia and Tartous, the most important citrus-producing areas in Syria. Each sample consisted of young leaves taken from four sides of each tree. The samples were tested for the presence of Citrus exocortis viroid (CEVd) and Citrus dwarf viroid (CVd-III). Total plant RNAs were extracted from leaves according to Foissac et al. (2001), and used as templates for reverse transcription (RT)-PCR using specific primers for both viroids. PCR products of the expected size (297 bp and 370 bp) for CVd-III and CEVd, respectively, were cloned and sequenced. Furthermore, previously infected budwood from five infected citrus trees were graft inoculated onto an Etrog citron (Citrus medica) indicator plants and maintained in a temperature controlled greenhouse. Biological indexing evidenced mild epinasty, leaf curling and typical stunting symptoms, confirming the presence of viroids. Results of RT-PCR showed that 24 out of 65 samples were infected by citrus viroids, including ten and eight that reacted positively to CEVd and CVd-III, respectively, and six that showed mixed infection by both viroids. The retrieved sequences were deposited in GenBank under accession numbers LN681197 and LN681196 for CVd-III and CEVd, respectively. To our knowledge, this is the first molecular identification of CEVd and CVd-III in citrus trees in Syria.

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.

A mutual titer-enhancing relationship and similar localization patterns between Citrus exocortis viroid and Hop stunt viroid co-infecting two citrus cultivars.

BackgroundCitrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd) are commonly found simultaneously infecting different citrus cultivars in Taiwan. A crucial question to be addressed is how accumulations of these two viroids affect each other in an infected plant. In this study, we investigated the relationship between the two viroids at macroscopic and microscopic levels.MethodsCEVd and HSVd titers were examined by real-time RT-PCR in 17 plants of two citrus cultivars (blood orange and Murcott mandarin) every 3 months (spring, summer, fall and winter) from 2011 to 2013. Three nonparametric tests (Spearman’s rank correlation coefficient, Kendall’s tau rank correlation coefficient and Hoeffding’s inequality) were performed to test the correlation between CEVd and HSVd. Cellular and subcellular localizations of the two viroids were detected by digoxigenin- and colloidal gold-labeled in situ hybridization using light and transmission electron microscopy.ResultsThe two viroids were unevenly distributed in four different types of citrus tissues (rootstock bark, roots, twig bark and leaves). Compared with blood orange, Murcott mandarin was generally more susceptible to CEVd and HSVd infection. Both viroids replicated and preferentially accumulated in the underground tissues of the two citrus cultivars. Except for blood orange at high temperatures, significant positive correlations were observed between the two viroids in specific tissues of both cultivars. Relative to concentrations under single-infection conditions, the CEVd population significantly increased under double infection during half of the 12 monitored seasons; in contrast, the population of HSVd significantly increased under double infection during only one season. At cellular/subcellular levels, the two viroids showed similar localization patterns in four tissues and the cells of these tissues in the two citrus cultivars.ConclusionsOur findings of titer enhancement, localization similarity, and lack of symptom aggravation under CEVd and HSVd double infection suggest that the two viroids have a positive relationship in citrus. The combination of molecular and cellular techniques used in this study provided evidence of titer correlation and localization of co-infecting viroids in the host. These methods may thus be useful tools for exploring viroid–viroid and viroid–host interactions.

Novel variants of citrus exocortis viroid from southern of Iran

Novel variants of citrus exocortis viroid from southern of Iran

GRAPEVINE VIROIDS AND GRAPEVINE FANLEAF VIRUS IN NORTH- WEST IRAN

Grapevine-infecting viroids do not induce symptoms, except for Grapevine yellow speckle viroid-1 (GYSVd-1) and Grapevine yellow speckle viroid-2 (GYSVd-2), the agents of yellow speckle (YS), a disease characterized by yellow spots or flecks scattered on the leaf blade. The association of these viroids with Grapevine fanleaf virus (GFLV) is thought to elicit vein banding (VB), a syndrome characterized by chrome yellow flecks localized along the main veins and progressing into the interveinal areas of affected vines. The occurrence of these diseases and their causal agents was investigated in north-west Iran with a survey in which 137 vines were tested by multiplex RT-PCR for the presence of the five known grapevines viroids. GYSVd-1, GYSVd-2, Australian grapevine viroid (AGVd) and Hop stunt viroid (HSVd) were detected in 91%, 64%, 95%, and 100% of the tested samples, respectively, whereas Citrus exocortis viroid (CEVd) was not found. Combinations of three and four different viroids were present in most plants (88%) whereas GFLV was found in 50 samples (37%). The Iranian isolates of GYSVd-1, GYSVd-2, HSVd and AGVd showed minor molecular changes compared with the respective reference strains from grapevine. VB occurred in 22 vines infected by GYSVd-1, GYSVd-2 and GFLV, whereas YS symptoms, which occurred in 10% of the tested plants, were always shown by vines infected by GYSVd-1 and/or GYSVd-2. These findings are in line with the notion that assigns to GYSVd-1 and GYSVd-2 a role in the induction of YS and to both viroids and GFLV the genesis of VB.

An assessment of the transmission rate of four pospiviroid species through tomato seeds

Greenhouse experiments were conducted to investigate the importance of tomato seeds as a route of pospiviroid dissemination. More than 7000 seeds were collected from tomato plants mechanically inoculated with Tomato apical stunt viroid, Citrus exocortis viroid, Columnea latent viroid and Potato spindle tuber viroid. Fruits and part of the seeds were found to be 100 % infected by the inoculated pospiviroid by means of RT-PCR testing. Much of the rest of the seeds were sown and produced about 4700 seedlings. None of the seedlings were found positive for the respective pospiviroid. This shows that pospiviroid seed transmission in tomato is very rare.

Phloem restriction of viroids in three citrus hosts is overcome by grafting with Etrog citron: potential involvement of a translocatable factor.

Viroid systemic spread involves cell-to-cell movement from initially infected cells via plasmodesmata, long-distance movement within the phloem and again cell-to-cell movement to invade distal tissues including the mesophyll. Citrus exocortis viroid (CEVd), hop stunt viroid, citrus bent leaf viroid, citrus dwarfing viroid, citrus bark cracking viroid and citrus viroid V remained phloem restricted when singly infecting Citrus karna, Citrus aurantium and Poncirus trifoliata, but not Etrog citron, where they were additionally detected in mesophyll protoplasts. However, when CEVd-infected C. karna was side-grafted with Etrog citron--with the resulting plants being composed of a C. karna stock and an Etrog citron branch--the viroid was detected in mesophyll protoplasts of the former, thus indicating that the ability of Etrog citron to support viroid invasion of non-vascular tissues was transferred to the stock. Further results suggest that a translocatable factor from Etrog citron mediates this viroid trafficking.

Viroidler ve Türkiye'de Saptanan Viroid Hastalık Etmenleri

Viroids are circular, single-stranded RNAs that infect plants. Viroids belong to two families, Pospiviroidae and Avsunviroidae, whose members replicate in the nucleus and chloroplast of plant cells, respectively. The host range of viroids includes vegetable crops, ornamentals, and woody perennials, depending upon the viroid species. Viroids are easily transmitted mechanically through contaminated pruning tools. They can also be spread vegetatively by grafting, tuber propagation, infected seed, pollen, and insects. There are over 30 known viroid species that are taxonomically divided into two families. Viroids are also responsible for several cultivated plant diseases in Turkey. Viroids and viroid disease agents causing plant disease (Potato spindle tuber viroid, Chrysanthemum stunt viroid, Citrus exocortis viroid, Hop stunt viroid, Citrus cachexia viroid, Citrus viroid III–IV, Grapevine yellow speckle viroid 1–2, Avocado sunblotch viroid, Peach latent mosaic viroid) in Turkey were discussed in this review

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.