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Vicente Pallas - One of the best experts on this subject based on the ideXlab platform.
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first detection of grapevine rupestris stem pitting associated virus and grapevine rupestris vein feathering virus and new phylogenetic groups for grapevine fleck virus and Hop Stunt Viroid isolates revealed from grapevine field surveys in spain
Phytopathologia Mediterranea, 2016Co-Authors: Nicola Fiore, Vicente Pallas, Alan Zamorano, Nicolas Sanchezdiana, Ximena Gonzalez, J A SancheznavarroAbstract:Evaluation of the prevalence of virus and Viroid infections was conducted in a grapevine field collection in Valencia, Spain. Samples of autochthonous and traditional grapevine cultivars were collected during November 2011 and tested for the presence of fourteen viruses and five Viroids, using RT-PCR. The prevalent viruses were Grapevine rupestris stem pitting-associated virus (GRSPaV: 49% infected samples) and Grapevine leafroll-associated virus 2 (GLRaV-2: 15% of samples). GLRaV-1, GLRaV-3, GLRaV-4 (variants 4 and 5), Grapevine fanleaf virus , Grapevine fleck virus (GFkV), Grapevine rupestris vein feathering virus (GRVFV) and Grapevine virus A were also detected. Hop Stunt Viroid (HSVd: 92% of plants infected) and Grapevine yellow speckle Viroid 1 (6% of plants) were also detected. Mixed infections with two, and up to six different viruses and/or Viroids were common. Only five samples (4%) were free from 19 pathogens tested. This is the first report of GLRaV-4 (variants 4 and 5) in the Valencia region of Spain, and the first record of GRSPaV and GRVFV in this country. Phylogenetic analyses performed with the sequences of these viruses showed that the Spanish isolates of GLRaV-4, GFkV and HSVd belong to new phylogenetic groups.
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alterations in host dna methylation in response to constitutive expression of Hop Stunt Viroid rna in nicotiana benthamiana plants
Plant Pathology, 2015Co-Authors: Mayte Castellano, German Martinez, Vicente Pallas, Gustavo GomezAbstract:Increasing evidence points to the epigenetic regulation of transcriptional activity as a general mechanism controlling host response to pathogen infection. Recent results revealed that cucumber plants infected with Hop Stunt Viroid (HSVd) showed alterations in the DNA methylation pattern of usually silenced rRNA genes that are consequently transcriptionally reactivated during infection. These results indicated that, at least in cucumber, HSVd pathogenesis could be associated with disruptions in the transcriptional mechanisms regulated by epigenetic modifications of host DNA. To determine if this disorder in host regulatory activity could be a common phenomenon that occurs in other Viroid–host combinations, here, the transcriptional activity in transgenic Nicotiana benthamiana constitutively expressing HSVd (HSVd/Nb) was analysed. It was observed that the HSVd RNA accumulation in N. benthamiana mimics the alteration induced by Viroid infection in cucumber, promoting the accumulation of ribosomal-derived small RNAs (rb-sRNAs). Bisulphite sequencing demonstrated that this phenomenon was linked to a loss of symmetric cytosine methylation and correlated with an increasing accumulation of rRNA precursors in HSVd/Nb plants. The data supports the hypothesis that the interference of this pathogenic long noncoding RNA (lncRNA) in epigenetic mechanisms (associated to rDNA methylation) controlling plant gene expression is not restricted to a specific host. Thus, this phenomenon may be more general and able to occur in other Viroid–plant interactions.
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high throughput sequencing of Hop Stunt Viroid derived small rnas from cucumber leaves and phloem
Molecular Plant Pathology, 2010Co-Authors: German Martinez, Vicente Pallas, Livia Donaire, Cesar Llave, Gustavo GomezAbstract:SUMMARY Small RNA (sRNA)-guided processes, referred to as RNA silencing, regulate endogenous and exogenous gene expression. In plants and some animals, these processes are noncell autonomous and can operate beyond the site of initiation. Viroids, the smallest self-replicating plant pathogens known, are inducers, targets and evaders of this regulatory mechanism and, consequently, the presence of Viroid-derived sRNAs (vd-sRNAs) is usually associated with Viroid infection. However, the pathways involved in the biogenesis of vd-sRNAs are largely unknown. Here, we analyse, by high-throughput pyrosequencing, the profiling of the Hop Stunt Viroid (HSVd) vd-sRNAs recovered from the leaves and phloem of infected cucumber (Cucumis sativus) plants. HSVd vd-sRNAs are mostly 21 and 22 nucleotides in length and derived equally from plus and minus HSVd RNA strands. The widespread distribution of vd-sRNAs across the genome reveals that the totality of the HSVd RNA genome contributes to the formation of vd-sRNAs. Our sequence data suggest that Viroid-derived double-stranded RNA functions as one of the main precursors of vd-sRNAs. Remarkably, phloem vd-sRNAs accumulated preferentially as 22-nucleotide species with a consensus sequence over-represented. This bias in size and sequence in the HSVd vd-sRNA population recovered from phloem exudate suggests the existence of a selective trafficking of vd-sRNAs to the phloem tissue of infected cucumber plants.
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High‐throughput sequencing of Hop Stunt Viroid‐derived small RNAs from cucumber leaves and phloem
Molecular Plant Pathology, 2010Co-Authors: German Martinez, Vicente Pallas, Livia Donaire, Cesar Llave, Gustavo GomezAbstract:SUMMARY Small RNA (sRNA)-guided processes, referred to as RNA silencing, regulate endogenous and exogenous gene expression. In plants and some animals, these processes are noncell autonomous and can operate beyond the site of initiation. Viroids, the smallest self-replicating plant pathogens known, are inducers, targets and evaders of this regulatory mechanism and, consequently, the presence of Viroid-derived sRNAs (vd-sRNAs) is usually associated with Viroid infection. However, the pathways involved in the biogenesis of vd-sRNAs are largely unknown. Here, we analyse, by high-throughput pyrosequencing, the profiling of the Hop Stunt Viroid (HSVd) vd-sRNAs recovered from the leaves and phloem of infected cucumber (Cucumis sativus) plants. HSVd vd-sRNAs are mostly 21 and 22 nucleotides in length and derived equally from plus and minus HSVd RNA strands. The widespread distribution of vd-sRNAs across the genome reveals that the totality of the HSVd RNA genome contributes to the formation of vd-sRNAs. Our sequence data suggest that Viroid-derived double-stranded RNA functions as one of the main precursors of vd-sRNAs. Remarkably, phloem vd-sRNAs accumulated preferentially as 22-nucleotide species with a consensus sequence over-represented. This bias in size and sequence in the HSVd vd-sRNA population recovered from phloem exudate suggests the existence of a selective trafficking of vd-sRNAs to the phloem tissue of infected cucumber plants.
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analysis of symptoms developed in nicotiana benthamiana plants expressing dimeric forms of Hop Stunt Viroid
Journal of Plant Pathology, 2008Co-Authors: German Martinez, Vicente Pallas, Gustavo GomezAbstract:Viroids are sub-viral plant pathogens consisting of covalently closed circular RNAs that infect herbaceous and woody hosts. They do not code for any peptide or protein and therefore are fully dependent on the host machinery for most biological processes, including pathogenesis. Certain Viroid-host combinations show severe symptoms but the biochemical bases of this process are currently unknown. In this work, we have characterized the symptoms induced by Hop Stunt Viroid (HSVd) when provided as a transgene in Nicotiana benthamiana plants, a natural non-host that is able to process and translocate HSVd. The presence of HSVd in this transgenic plant causes severe Stunting and decrease in the leaf size, followed by strong alteration of flower development and reduced fertility. These symptoms are similar to those induced by HSVd in natural infections, indicating that in N. benthamiana HSVd expression mimics the metabolic disorders induced in natural hosts. Therefore, these transgenic plants may be useful to study the Viroid-host interactions leading to pathogenesis.
Gustavo Gomez - One of the best experts on this subject based on the ideXlab platform.
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alterations in host dna methylation in response to constitutive expression of Hop Stunt Viroid rna in nicotiana benthamiana plants
Plant Pathology, 2015Co-Authors: Mayte Castellano, German Martinez, Vicente Pallas, Gustavo GomezAbstract:Increasing evidence points to the epigenetic regulation of transcriptional activity as a general mechanism controlling host response to pathogen infection. Recent results revealed that cucumber plants infected with Hop Stunt Viroid (HSVd) showed alterations in the DNA methylation pattern of usually silenced rRNA genes that are consequently transcriptionally reactivated during infection. These results indicated that, at least in cucumber, HSVd pathogenesis could be associated with disruptions in the transcriptional mechanisms regulated by epigenetic modifications of host DNA. To determine if this disorder in host regulatory activity could be a common phenomenon that occurs in other Viroid–host combinations, here, the transcriptional activity in transgenic Nicotiana benthamiana constitutively expressing HSVd (HSVd/Nb) was analysed. It was observed that the HSVd RNA accumulation in N. benthamiana mimics the alteration induced by Viroid infection in cucumber, promoting the accumulation of ribosomal-derived small RNAs (rb-sRNAs). Bisulphite sequencing demonstrated that this phenomenon was linked to a loss of symmetric cytosine methylation and correlated with an increasing accumulation of rRNA precursors in HSVd/Nb plants. The data supports the hypothesis that the interference of this pathogenic long noncoding RNA (lncRNA) in epigenetic mechanisms (associated to rDNA methylation) controlling plant gene expression is not restricted to a specific host. Thus, this phenomenon may be more general and able to occur in other Viroid–plant interactions.
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High‐throughput sequencing of Hop Stunt Viroid‐derived small RNAs from cucumber leaves and phloem
Molecular Plant Pathology, 2010Co-Authors: German Martinez, Vicente Pallas, Livia Donaire, Cesar Llave, Gustavo GomezAbstract:SUMMARY Small RNA (sRNA)-guided processes, referred to as RNA silencing, regulate endogenous and exogenous gene expression. In plants and some animals, these processes are noncell autonomous and can operate beyond the site of initiation. Viroids, the smallest self-replicating plant pathogens known, are inducers, targets and evaders of this regulatory mechanism and, consequently, the presence of Viroid-derived sRNAs (vd-sRNAs) is usually associated with Viroid infection. However, the pathways involved in the biogenesis of vd-sRNAs are largely unknown. Here, we analyse, by high-throughput pyrosequencing, the profiling of the Hop Stunt Viroid (HSVd) vd-sRNAs recovered from the leaves and phloem of infected cucumber (Cucumis sativus) plants. HSVd vd-sRNAs are mostly 21 and 22 nucleotides in length and derived equally from plus and minus HSVd RNA strands. The widespread distribution of vd-sRNAs across the genome reveals that the totality of the HSVd RNA genome contributes to the formation of vd-sRNAs. Our sequence data suggest that Viroid-derived double-stranded RNA functions as one of the main precursors of vd-sRNAs. Remarkably, phloem vd-sRNAs accumulated preferentially as 22-nucleotide species with a consensus sequence over-represented. This bias in size and sequence in the HSVd vd-sRNA population recovered from phloem exudate suggests the existence of a selective trafficking of vd-sRNAs to the phloem tissue of infected cucumber plants.
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high throughput sequencing of Hop Stunt Viroid derived small rnas from cucumber leaves and phloem
Molecular Plant Pathology, 2010Co-Authors: German Martinez, Vicente Pallas, Livia Donaire, Cesar Llave, Gustavo GomezAbstract:SUMMARY Small RNA (sRNA)-guided processes, referred to as RNA silencing, regulate endogenous and exogenous gene expression. In plants and some animals, these processes are noncell autonomous and can operate beyond the site of initiation. Viroids, the smallest self-replicating plant pathogens known, are inducers, targets and evaders of this regulatory mechanism and, consequently, the presence of Viroid-derived sRNAs (vd-sRNAs) is usually associated with Viroid infection. However, the pathways involved in the biogenesis of vd-sRNAs are largely unknown. Here, we analyse, by high-throughput pyrosequencing, the profiling of the Hop Stunt Viroid (HSVd) vd-sRNAs recovered from the leaves and phloem of infected cucumber (Cucumis sativus) plants. HSVd vd-sRNAs are mostly 21 and 22 nucleotides in length and derived equally from plus and minus HSVd RNA strands. The widespread distribution of vd-sRNAs across the genome reveals that the totality of the HSVd RNA genome contributes to the formation of vd-sRNAs. Our sequence data suggest that Viroid-derived double-stranded RNA functions as one of the main precursors of vd-sRNAs. Remarkably, phloem vd-sRNAs accumulated preferentially as 22-nucleotide species with a consensus sequence over-represented. This bias in size and sequence in the HSVd vd-sRNA population recovered from phloem exudate suggests the existence of a selective trafficking of vd-sRNAs to the phloem tissue of infected cucumber plants.
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analysis of symptoms developed in nicotiana benthamiana plants expressing dimeric forms of Hop Stunt Viroid
Journal of Plant Pathology, 2008Co-Authors: German Martinez, Vicente Pallas, Gustavo GomezAbstract:Viroids are sub-viral plant pathogens consisting of covalently closed circular RNAs that infect herbaceous and woody hosts. They do not code for any peptide or protein and therefore are fully dependent on the host machinery for most biological processes, including pathogenesis. Certain Viroid-host combinations show severe symptoms but the biochemical bases of this process are currently unknown. In this work, we have characterized the symptoms induced by Hop Stunt Viroid (HSVd) when provided as a transgene in Nicotiana benthamiana plants, a natural non-host that is able to process and translocate HSVd. The presence of HSVd in this transgenic plant causes severe Stunting and decrease in the leaf size, followed by strong alteration of flower development and reduced fertility. These symptoms are similar to those induced by HSVd in natural infections, indicating that in N. benthamiana HSVd expression mimics the metabolic disorders induced in natural hosts. Therefore, these transgenic plants may be useful to study the Viroid-host interactions leading to pathogenesis.
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incidence and genetic diversity of peach latent mosaic Viroid and Hop Stunt Viroid in stone fruits in serbia
European Journal of Plant Pathology, 2008Co-Authors: B Mandic, Gustavo Gomez, A Myrta, Al M Rwahnih, Vicente PallasAbstract:Tissue-imprint hybridization (TIH) assay was validated for large-scale detection of Peach latent mosaic Viroid (PLMVd) and Hop Stunt Viroid (HSVd). All 72 collected leaves (100%) from 2 PLMVd- and 2 HSVd-infected trees were positive in TIH, regardless of the geographic orientation of the scaffold, level of the canopy and position of the leaf in the shoot. In a large-scale survey in Serbia, we tested by TIH 871 trees of stone fruits, representing 602 cultivars from fruit collections in Belgrade, Cacak and Novi Sad. PLMVd was detected in 185 (50%) peach trees or 95 (54%) cultivars and HSVd in 2 apricot trees and cultivars (2%). The occurrence of HSVd is a new report for Serbia. No Viroid infection was found in European plums, sweet cherries, sour cherries and wild Prunus spp. PLMVd-infected peach cultivars originated from the world’s main breeding centres of this crop. Western European and Asian cultivars were the most infected (58%) followed by those originating from North America (50%). Nine PLMVd and two HSVd isolates were sequenced and analyzed. All showed PMLVd sequences clustered together in the previously reported phylogenetic group III. Both HSVd isolates were found to be derived from recombinant events, but that of the cv. Saturn represented a putative new phylogenetic group of HSVd.
G R G Clover - One of the best experts on this subject based on the ideXlab platform.
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first report of grapevine yellow speckle Viroid 1 and Hop Stunt Viroid in grapevine vitis vinifera in new zealand
Plant Disease, 2011Co-Authors: L I Ward, G M Burnip, Lia W Liefting, S J Harper, G R G CloverAbstract:In February 2009, grapevines (Vitis vinifera) in a commercial vineyard in Auckland were showing shortened, spindly canes with tiny leaves. Approximately 10% of the vines were affected. An RNeasy Plant Mini Kit (Qiagen, Valencia, CA) was used to isolate total RNA from leaves collected from six symptomatic (cvs. BAC0022A and Syrah) and eight symptomless vines (cvs. BAC0022A, Syrah, and Chardonnay). RNA was tested by reverse transcription-PCR for the presence of Australian grapevine Viroid, Citrus exocortis Viroid, Grapevine yellow speckle Viroid 1 (GYSVd-1), Grapevine yellow speckle Viroid 2, and Hop Stunt Viroid (HSVd). Four of the six symptomatic and all the symptomless vines tested positive for GYSVd-1 using primers 5′-TGTGGTTCCTGTGGTTTCAC-3′ and 5′-ACCACAAGCAAGAAGATCCG-3′, which amplify the complete genome (368 bp), and published primers PBCVd100C/194H (3), which amplify a 220-bp region of the genome. Amplicons from each PCR were transformed into a pCR 4-TOPO vector (Invitrogen, Carlsbad, CA), cloned, a...
Olufemi J Alabi - One of the best experts on this subject based on the ideXlab platform.
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first report of grapevine yellow speckle Viroid 1 grapevine yellow speckle Viroid 2 and Hop Stunt Viroid infecting grapevines vitis spp in nigeria
Plant Disease, 2018Co-Authors: A M Zongoma, Al M Rwahnih, D B Dangora, S P Bako, M D Alegbejo, Olufemi J AlabiAbstract:Grapevine yellow speckle Viroid 1 (GYSVd-1), Grapevine yellow speckle Viroid 2 (GYSVd-2; genus ApscaViroid), and Hop Stunt Viroid (HSVd; genus HostuViroid) are three of six Viroid species documented globally from grapevines (Di Serio et al. 2017). These small plant-pathogenic, non-protein coding, circular RNAs may or may not elicit disease symptoms in grapevines depending on vagaries of the environment and their presence in mixed infection with other grapevine-infecting viruses. During 2016, surveys were conducted in the Northern Guinea Savannah agroecological zone of Nigeria to document the occurrence of grapevine viruses and Viroids. A total of 318 leaf tissue samples belonging to five cultivars were collected during the survey across 28 vineyards. The samples were dried under CaCl₂ at room temperature, and then shipped under USDA-APHIS-PPQ permit to Texas AM MF576417–428) shared 96 to 100% identity with several HSVd GenBank isolates. The complete GYSVd-1 RNA derived in this study (365 to 367 nt; MF576399–407) shared 96 to 99% identity with several GYSVd-1 GenBank isolates. The complete GYSVd-2 RNA derived in this study (361 nt; MF576408–416) shared 97 to 99% identity with several GYSVD-2 GenBank isolates. No discernible symptoms were associated with the presence of all three Viroid species in their source grapevines. To our knowledge, this is the first report of Viroids infecting grapevine in Nigeria. Studies are ongoing to further elucidate the prevalence of all three Viroids in Nigerian vineyards and their occurrences in mixed infections with other graft-transmissible agents of grapevine.
T H Hung - One of the best experts on this subject based on the ideXlab platform.
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a mutual titer enhancing relationship and similar localization patterns between citrus exocortis Viroid and Hop Stunt Viroid co infecting two citrus cultivars
Virology Journal, 2015Co-Authors: Mengling Wu, Tanglong Shen, T H HungAbstract:Citrus 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. CEVd 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. The 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. Our 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.
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multiplex detection distribution and genetic diversity of Hop Stunt Viroid and citrus exocortis Viroid infecting citrus in taiwan
Virology Journal, 2015Co-Authors: Mengling Wu, Tanglong Shen, T H HungAbstract:Background Two 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.
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Multiplex detection, distribution, and genetic diversity of Hop Stunt Viroid and Citrus exocortis Viroid infecting citrus in Taiwan
Virology Journal, 2015Co-Authors: Mengling Wu, Tanglong Shen, T H HungAbstract:Background Two 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. Methods Multiplex 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. Results HSVd 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. Conclusions Moderately 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.