Citrus Exocortis Viroid Infection Research Articles

Ethylene is Involved in Symptom Development and Ribosomal Stress of Tomato Plants upon Citrus Exocortis Viroid Infection.

Citrus exocortis viroid (CEVd) is known to cause different symptoms in citrus trees, and its mechanism of infection has been studied in tomato as an experimental host, producing ribosomal stress on these plants. Some of the symptoms caused by CEVd in tomato plants resemble those produced by the phytohormone ethylene. The present study is focused on elucidating the relationship between CEVd infection and ethylene on disease development. To this purpose, the ethylene insensitive Never ripe (Nr) tomato mutants were infected with CEVd, and several aspects such as susceptibility to infection, defensive response, ethylene biosynthesis and ribosomal stress were studied. Phenotypic characterization revealed higher susceptibility to CEVd in these mutants, which correlated with higher expression levels of both defense and ethylene biosynthesis genes, as well as the ribosomal stress marker SlNAC082. In addition, Northern blotting revealed compromised ribosome biogenesis in all CEVd infected plants, particularly in Nr mutants. Our results indicate a higher ethylene biosynthesis in Nr mutants and suggest an important role of this phytohormone in disease development and ribosomal stress caused by viroid infection.

Global Transcriptomic Analysis Reveals Insights into the Response of 'Etrog' Citron (Citrus medica L.) to Citrus Exocortis Viroid Infection.

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.

Essential oil components of Citrus cultivar‘MALTAISE DEMI SANGUINE’ (Citrus sinensis) as affected by the effects of rootstocks and viroid infection

ABSTRACT ‘Maltaise demi sanguine’ cultivars are usually grafted on sour orange rootstock. Nonetheless, this rootstock is highly susceptible to Citrus tristeza virus (CTV). A trial was set up in order to present new rootstocks as an alternative to sour orange. The effects of eight citrus rootstocks upon the volatile flavor components of the “Maltaise demi sanguine” peel were investigated in both healthy plants and those inoculated with Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd). Rootstocks and viroid infection had no significant effect on essential oil yields. Oil components were analyzed by GC-MS. Total of 69 compounds were identified, representing 99% of the total oil. Limonene was the major component in all samples (59.53–95.34%). Principal components and hierarchical cluster analyses revealed that essential oils from plants grafted on Alemow and Carrizo citrange showed the most variable compositions in cases of healthy plants and those inoculated with CEVd. For those plants infected by HSVd, Cleopatra mandarin seemed to be the most affected. Overall, the combined effect of CEVd infection and the use of rootstocks Alemow, Carrizo citrange, and Volkamer lemon had the most pronounced chemical variability.

Innate Immunity Activation and RNAi Interplay in Citrus Exocortis Viroid-Tomato Pathosystem.

Although viroids are the smallest and simplest plant pathogens known, the molecular mechanisms underlying their pathogenesis remain unclear. To unravel these mechanisms, a dual approach was implemented consisting of in silico identification of potential tomato silencing targets of pospiviroids, and the experimental validation of these targets through the sequencing of small RNAs and RNA ends extracted from tomatoes infected with a severe isolate of Citrus exocortis viroid (CEVd). The generated RNA ends were also used to monitor the differentially-expressed genes. These analyses showed that when CEVd symptoms are well established: (i) CEVd are degraded by at least three Dicer-like (DCL) proteins and possibly by RNA-induced silencing complex (RISC), (ii) five different mRNAs are partially degraded through post-transcriptional gene silencing (PTGS), including argonaute 2a, which is further degraded in phasiRNAs, (iii) Dicer-like 2b and 2d are both upregulated and degraded in phasiRNAs, and (iv) CEVd infection induced a significant shift in gene expression allowing to explain the usual symptoms of pospiviroids on tomato and to demonstrate the constant activation of host innate immunity and systemic acquired resistance (SAR) by these pathogenic RNAs. Finally, based on in silico analysis, potential immunity receptor candidates of viroid-derived RNAs are suggested.

A noncoding plant pathogen provokes both transcriptional and posttranscriptional alterations in tomato

Viroids are single-stranded, circular, noncoding RNAs that infect plants, causing devastating diseases. In this work, we employed 2D DIGE, followed by MS identification, to analyze the response of tomato plants infected by Citrus exocortis viroid (CEVd). Among the differentially expressed proteins detected, 45 were successfully identified and classified into different functional categories. Validation results by RT-PCR allowed us to classify the proteins into two expression groups. First group included genes with changes at the transcriptional level upon CEVd infection, such as an endochitinase, a β-glucanase, and pathogenesis-related proteins, PR10 and P69G. All these defense proteins were also induced by gentisic acid, a pathogen-induced signal in compatible interactions. The second group of proteins showed no changes at the transcriptional level and included several ribosomal proteins and translation factors, such as the elongation factors 1 and 2 and the translation initiation factor 5-alpha. These results were validated by 2D Western blot, and possible PTMs caused by CEVd infection were detected. Moreover, an interaction between eukaryotic elongation factor 1 and CEVd was observed by 2D Northwestern. The present study provides new protein-related information on the mechanisms of plant resistance to pathogens.

First Report of Citrus exocortis viroid in a Verbena sp. in Montenegro.

Numerous ornamental plants have been found to be symptomless hosts of various pospiviroids including Citrus exocortis viroid (CEVd), and hence, may serve as potential inoculum reservoirs for susceptible vegetable plants. Production of tomato, potato, and pepper, on which viroids from the genus Pospiviroid can cause severe damage, represents two-thirds of the vegetable production in Montenegro. We tested vegetable, ornamental, and weed host plants for the presence of pospiviroids in September 2011. Altogether 80 samples were taken. Samples of ornamental plants (15 of Petunia spp., 7 of Impatiens spp., 4 of Verbena spp., 3 of Dahlia spp., 3 of Pittosporum tobira, 3 of Vinca spp., 2 of Brugmansia spp., 2 of avocado, 2 of Portulaca spp., and 1 of Datura sp.) were taken from three places of production. One sample per species was collected from symptomless eggplants, tomatoes, sweet peppers, and avocados in the vicinity of one glasshouse with ornamental plants. Twenty-two samples from sweet pepper and seven samples from tomato, all grown under cover and all showing potential virus-like symptoms, were collected from three places of vegetable production. Two samples of Solanum nigrum and three samples of unidentified weed species belonging to genus Solanum were taken from two glasshouses. With the exception of weed plants, samples consisted of fully developed leaves collected from five plants. All sampled ornamental and weed plants were symptomless. RNA was extracted from approximately 15 mg of leaf tissue with the MagMAX-96 Total RNA Isolation Kit (Life Technologies, Foster City, CA) in accordance with the manufacturer's instructions for the MagMAX Express Magnetic Particle Processor. Samples were tested by reverse transcription (RT)-PCR using semi-universal pospiviroid primers (Pospi1-RE/FW and Vid-RE/FW [3]). None of the samples reacted with the Vid-RE/FW primer pair. An amplicon of an expected size (approximately 196 nt) was produced with the Pospi1-RE/FW primer pair from one Verbena sp. sample. Direct sequencing was performed by Macrogen (Amsterdam, The Netherlands). Sequence analysis indicated the presence of CEVd. This finding was confirmed by sequence analysis of the DNA product obtained by RT-PCR using Pospi1-FW/RE from a new extraction. Further analyses using primer pairs CEVd-AS/S (1) and CEVd-FW2/RE2 (4) were performed to obtain the full viroid sequence. The sequence of 372 nt was deposited in GenBank (Accession No. JN872140) and had 99% identity with two CEVd sequences from Verbena spp. (Accession Nos. EF192396 and DQ094297). To our knowledge, this is the first report of CEVd in a Verbena sp. in Montenegro and the second report in Europe (4). CEVd has been detected in Verbena spp. also in India and Canada and can be transmitted by seed (2). The infected Verbena sp. plants were not destroyed, since CEVd is not listed as a quarantine organism in Montenegro. The spread of CEVd infection to tomato could devastate the production of this crop in Montenegro.

Citrus exocortis viroid infecting twenty year old Solanum jasminoides plants in Italy.

Potato spindle tuber viroid (PSTVd), the type species of the genus Pospiviroid, and other members of the same genus, including Citrus exocortis viroid (CEVd), have been found in symptomless ornamental solanaceous plants, which may serve as potential inoculum for susceptible horticultural crops, as shown in Italy by a recent PSTVd outbreak in tomato (Navarro et al., 2009). We have recently identified CEVd in Solanum jasminoides in Apulia (southern Italy), an ornamental species in which this viroid is widely distributed, especially in the nurseries (Torchetti et al., 2011). Here we report the identification of CEVd in a more than 20-year-old S. jasminoides plant from Latium (central Italy). The viroid was first detected by RT-PCR using generic primers for pospiviroids (Bostan et al., 2004); then, it was identified by RT-PCR using CEVd-specific primers (Torchetti et al., 2011) followed by cDNA sequencing. The amplicon had a sequence 97-98% identical to CEVd variants previously reported from S. jasminoides in other countries (GenBank accession Nos AM920649; GU300810; HQ423166). Tested samples were collected in a private garden located in a very isolated area, in which no other potential natural CEVd inoculum (i.e. Citrus spp.) were found, supporting the possibility that the S. jasminoides plants were already infected when transplanted. This finding dates back by at least twenty years the presence of CEVd infection of S. jasminoides in Italy.

Characterization of small RNAs derived from Citrus exocortis viroid (CEVd) in infected tomato plants

In plants, RNA silencing provides an adaptive immune system that inactivates pathogenic nucleic acids, guided by 21–24-mer RNAs of pathogen origin. The characterization of pathogen-related small RNAs (sRNAs) is relevant to uncovering the strategies used by pathogens to evade host defense responses. Several groups have reported the detection of viroid-derived sRNAs during infections, although the origin of these sRNAs and their chemical characteristics were poorly understood. Here, we describe the in vivo cleavage of Citrus exocortis viroid (CEVd) RNA into sRNAs of 21–22 nt, that are phosphorylated at their 5′-end and methylated at 3′. Our studies suggested that the CEVd genomic RNA might be the predominant in vivo substrate for cleavage by Dicer-like enzyme(s), which preferentially targeted residues mainly located within the right-end domain of the viroid. Further analysis on the accumulation levels of specific miRNAs controlling major regulators of leaf development and the miRNA pathway and the levels of their target mRNAs provided evidence that the endogenous tomato miRNA pathway was not affected by CEVd infection.

Citrus Viroids: Symptom Expression and Effect on Vegetative Growth and Yield of Clementine Trees Grafted on Trifoliate Orange.

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, CVd-IV, 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.

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

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

Correlation between Ornithine Decarboxylase and Putrescine in Tomato Plants Infected by Citrus Exocortis Viroid or Treated with Ethephon.

We have investigated the arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17) activities and the levels of conjugated polyamines to explain the decrease of free putrescine level caused by citrus exocortis viroid (CEVd) and ethephon treatment in tomato (Lycopersicon esculentum Mill. cv Rutgers) plants (J.M. Belles, J. Carbonell, V. Conejero [1991] Plant Physiol 96: 1053-1059). This decrease correlates with a decrease in ODC activity in CEVd-infected or ethephon-treated plants; ADC activity was not altered. CEVd infection had no effect on polyamine conjugates, and ethephon produced a decrease in putrescine conjugates. Interference with ethylene action by silver ions prevented the decrease in ODC activity and in free and conjugated putrescine. It is suggested that changes in putrescine level after CEVd infection and ethephon treatment are regulated via ODC activity and that conjugation is not involved.

Suppression by Citrus Exocortis Viroid Infection of the Naturally Occurring Inhibitor of the Conversion of 1-aminocyclopropane-1-carboxylic Acid to Ethylene by Tomato Microsomes

Citrus exocortis viroid (CEVd) infection of tomato cell cultures suppresses the constitutive inhibitor which blocks the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene by tomato microsomes. The inhibitor is associated to microsomal membranes and is also found in the water soluble fraction co-isolated from the cells. The inhibitory effect is concentration dependent, heat stable and could be removed from solution by dialysis. Its possible relationship with regulation of the viroid-induced ethylene production is discussed.

C 7(P32) and C 6(P34) PR proteins induced in tomato leaves by citrus exocortis viroid infection are chitinases

Two chitinases induced in tomato leaves ( Lycopersicon esculentum Mill cv. Rutgers) by citrus exocortis viroid (CEV) infection were purified. Their molecular masses determined by sodium dodecyl sulfate-gel electrophoresis were 32 kDa and 34 kDa and by filtration through Sephadex G-100 were 23 kDa and 25 kDa, respectively. These chitinases (P32 and P34) have been shown to be identical to the tomato pathogenesis-related proteins C 7 and C 6. They were purified in three stages: ammonium sulphate fractionation, chitin affinity chromatography and CM-Sepharose chromatography. The characterization of P32, the major component of the CEV-induced chitinase activity, revealed a basic protein (pI, 8·5), with a low optimum pH of 5·2 for hydrolytic activity, a high thermal stability and resistance to proteolytic degradation. These tomato chitinases have been shown to be serologically related to the tobacco chitinases. Our results lend further support to the hypothesis that enhanced chitinase activity is a component of a general mechanism of response to pathogens and other stress agents.

The induction and accumulation of the pathogenesis-related P69 proteinase in tomato during citrus exocortis viroid infection and in response to chemical treatments

Citrus exocortis viroid (CEV) infection in tomato plants induces the production and accumulation of several pathogenesis-related (PR) proteins. One of the most abundant PR proteins, P69, has previously been found to be an alkaline proteinase. By using an antiserum raised against P69 we have followed the accumulation of this protein during viroid infection, as well as its induction by ethephon and salicylic acid. We have also followed its distribution in viroid infected plants. The results suggest that ethylene is a mediator of the induction of P69 proteinase by citrus exocortis viroid and that symptom severity is correlated with the accumulation and activity of this protein.

ACC Synthesis as the Activated Step Responsible for the Rise of Ethylene Production Accompanying Citrus Exocortis Viroid Infection in Tomato Plants

AbstractEthylene production was stimulated during the period when systemic symptoms appeared in tomato plants infected with citrus exocortis viroid (CEV).Neither methionine nor S‐adenosylmethionine increased ethylene production in leaf discs. In contrast, 1‐aminocyclopropane‐l‐carboxylic acid (ACC) stimulated ethylene production notably. Whether viroid infection acted upon ACC production, its conversion to ethylene, or both, was studied by determining the time course of the concentration of ACC and its in vivo production and conversion rates. During early symptoms, ACC synthesis increased and then remained steady during the development of symptoms, but no difference in the capacity of conversion of ACC to ethylene between healthy and CEV‐infected tissues was observed. This indicates that ethylene production in tomato leaves showing systemic symptoms to CEV is activated at the level of ACC production.

Changes in the Pathway of Ethylene Production Following Citrus Exocortis Viroid Infection in Tomato Plants

Changes in the Pathway of Ethylene Production Following Citrus Exocortis Viroid Infection in Tomato Plants

Antiviroid effects of ribavirin on citrus exocortis viroid infection in Gynura aurantiaca DC

Treatments of Gynura aurantiaca DC plants, infected with citrus exocortis viroid, with concentrations of ribavirin from 300 to 1400 mgl−1 reduced viroid levels and led to almost complete remission of symptoms on newly developed leaves, When applied to plants soon after inoculation, ribavirin prevented the establishment of the viroid.