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Citrus exocortis viroid (CEVd) and citrus bark cracking viroid (CBCVd) are two important viroids that infect citrus plants and frequently occur as mixed infections in orchards. However, the mechanism of antagonism between the two viroids in mixed infections remains unclear. The CEVd/CBCVd–citron system and small RNA sequencing (sRNA‐seq) were used to study the antagonism. When CBCVd was inoculated before CEVd, the CEVd titre was significantly reduced and the symptoms were attenuated. Viroid‐derived sRNAs (vd‐sRNAs) from CEVd and CBCVd were predominantly 21‐nucleotide (nt) and 22‐nt in length and had similar 5′ base biases. Homologous sequences of the two viroids in the terminal right (TR) region are rich in vd‐sRNAs, and the high frequency vd‐sRNAs selected from the CBCVd TR region can be used to degrade the transcripts of CEVd in vivo directly. These results suggest that RNA silencing may play an important role in the antagonism of the two viroids, thus deepening our understanding of the molecular interaction of long noncoding RNAs in woody plants. Citrus bark cracking viroid (CBCVd) suppresses the replication and attenuates the symptoms of citrus exocortis viroid (CEVd) on citron, probably by cleaving CEVd via small RNAs from the CBCVd terminal right region using host RNA silencing.

Citrus are natural hosts of five viroid species: Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus viroid III (CVd-III), and Citrus viroid IV (CVd-IV). CEVd and specific sequence variants of HSVd are the causal agents of the wellknown diseases of citrus, exocortis and cachexia. Other viroids have been found to induce different degrees of stunting. Since commercial citrus trees are commonly infected with mixtures of these viroids, only limited information is available on their effect in species other than Etrog citron. A field assay was conducted to establish the effect of each viroid on Commune clementine trees grafted on Pomeroy trifoliate orange. Infected trees were periodically monitored over a 12-year period (1990 to 2002) for symptom expression, growth, and fruit yield. Only CEVd caused bark scaling on the trifoliate orange rootstock and marked dwarfing, both characteristic of exocortis disease as initially described. In addition, very conspicuous bumps were observed in the wood of the rootstock after removing the bark. Only those HSVd variants, previously characterized as pathogenic in several cachexia-sensitive species, induced pits and gum deposits characteristic of this disease in the clementine scion. Bark cracking symptoms on the trifoliate orange rootstock were also observed. They were associated with CVd-IV, HSVd, or CEVd infection, but in the latter, they were only clearly observed in trees that showed mild scaling. Other abnormalities (deep pits, crests, and gummy pits) were not associated with viroid infection. No specific symptoms resulted from infection with CBLVd and CVd-III. HSVd, CVdIV, and CBLVd had little or no effect in growth and yield, whereas CEVd and CVd-III caused a significant reduction of growth and yield, which became more pronounced over time with CEVd infection. Yield reduction was associated mainly with loss of production of large fruits. In general, there was a good correlation between reduction in vegetative growth and yield.

A number of viroids can cause serious damage on Citrus spp. ranging from stunting, bark scaling, yellowing and epinasty of leaves over stem pitting and gumming. However, so far, they have never been found in Thailand. In recent years, the import of orange and lime fruits from China and Cambodia, respectively, increased, and holds a substantial risk of viroid introduction and spread in Thailand. Orange and lime fruit samples in 2013 and 2014 were screened for the presence of citrus exocortis viroid (CEVd), hop stunt viroid (HSVd), citrus bent leaf viroid (CBLVd) and citrus dwarfing viroid (CDVd; CVd-IIId) by means of specific RT-PCR methods. Only CBLVd and CDVd were detected, clearly generating the expected amplification bands of around 320 and 300 base pairs, respectively. The presence of CBLVd and CDVd was confirmed by amplicon sequencing and RNA secondary structure analysis. About 34.2 and 19.5% of 41 samples (around 2300 fruits) of the imported lime fruit were infected with CBLVd and CDVd, respectively. CBLVd was detected in 62.3% of the 77 samples (around 2000 fruits) from imported oranges, while CDVd was found in 75.3%. This result indicates that the incidence of both CBLVd and CDVd in the imported citrus fruits is quite high. In addition, both viroid diseases have not been reported in Thailand. However, lack of information on the actual status of both viroids leads to difficulties in determining their impact on the Thai citrus industry.

Preliminary transmission assays conducted under greenhouse conditions demonstrated that Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus viroid III (CVd-III), and Citrus viroid IV (CVd-IV) can be mechanically transmitted from citron to citron (Citrus medica) by a single slash with a knife blade. The impact of mechanical transmission of viroids by pruning and harvesting operations was also demonstrated in experimental and commercial field plots. Transmission efficiency under field conditions ranged from 4% in 'Nules' clementine to 10% in 'Navelina' sweet orange and 21% in 'Verna' lemon. Transmission efficiency varied only slightly with viroid and donor hosts. The impact of viroid transmission on tree height, canopy volume, and crop harvest was minimal. When the donor host was coinfected with several viroids, the viroids were not necessarily cotransmitted. Considerations regarding viroid transmission in other climates are discussed. Measures to control viroid spread in nurseries should be mandatory in certification programs.

Citrus plants are natural hosts of five viroid species and large numbers of sequence variants. In this paper a simple and sensitive one step multiplex RT-PCR protocol with an internal control was utilised to simultaneously detect and differentiate five citrus viroids: Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus viroid-III (CVd-III) and Citrus viroid-IV (CVd-IV). In addition, a micro and rapid total nucleic acid extraction method was developed and the protocol applied to evaluate the occurrence and distribution of citrus viroids in China.

Citrus exocortis viroid (CEVd) is the causal agent of citrus exocortis disease. We employed CEVd-infected ‘Etrog’ citron as a system to study the feedback regulation mechanism using transcriptome analysis in this study. Three months after CEVd infection, the transcriptome of fresh leaves was analyzed, and 1530 differentially expressed genes were detected. The replication of CEVd in citron induced upregulation of genes encoding key proteins that were involved in the RNA silencing pathway such as Dicer-like 2, RNA-dependent RNA polymerase 1, argonaute 2, argonaute 7, and silencing defective 3, as well as those genes encoding proteins that are related to basic defense responses. Many genes involved in secondary metabolite biosynthesis and chitinase activity were upregulated, whereas other genes related to cell wall and phytohormone signal transduction were downregulated. Moreover, genes encoding disease resistance proteins, pathogenicity-related proteins, and heat shock cognate 70 kDa proteins were also upregulated in response to CEVd infection. These results suggest that basic defense and RNA silencing mechanisms are activated by CEVd infection, and this information improves our understanding of the pathogenesis of viroids in woody plants.

The credibility of a pathogen detection assay is measured using specific parameters including repeatability, specificity, sensitivity, and reproducibility. The use of high-throughput sequencing (HTS) as a routine detection assay for viruses and viroids in citrus was previously evaluated and, in this study, the reproducibility and sensitivity of the HTS assay were assessed. To evaluate the reproducibility of HTS, the same plants assayed in a previous study were sampled again, one year later, and assessed in triplicate using the same analyses to construct the virome profile. The sensitivity of the HTS assay was compared to routinely used RT-PCR assays in a time course experiment, to compensate for natural pathogen accumulation in plants over time. The HTS pipeline applied in this study produced reproducible and comparable results to standard RT-PCR assays for the detection of CTV and three viroid species in citrus. Even though the limit of detection of HTS can be influenced by pathogen concentration, sample processing method and sequencing depth, detection with HTS was found to be either equivalent or more sensitive than RT-PCR in this study.

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.

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 ‘Sécile’ 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 pépins’) 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.