Detection of Viroids by sPAGE Gel Electrophoresis.

Nucleic acid polyacrylamide gel electrophoresis (PAGE) has played a critical role in the identification and characterization of viroid RNAs. In addition, double PAGE has been a very efficient tool for the detection of viroids as it is sequence independent and is based on the viroid structure of covalently closed/circular molecules. sPAGE has been widely used for the identification of new viroids as well as a routine detection tool.

Viroids as Companions of a Professional Career.

Since the early 1970s when "virus-like" agents were considered as the cause of two diseases (potato spindle tuber and citrus exocortis), their study and further characterization have been linked to the development and use of molecular biology tools. Sucrose density gradient centrifugation and polyacrylamide gel electrophoresis (PAGE) played a critical role in the pioneering studies of PSTVd and citrus exocortis viroid (CEVd). This was later modified by using other PAGEs (sequential PAGE, return PAGE, two-dimensional PAGE), and/or different staining methods (ethidium bromide, silver nitrate, etc.). Since then, disease-causing agents suspected to be viroids were usually subjected to a number of tests to define their: (i) Molecular nature (RNA or DNA; single stranded or double stranded; circular or linear RNA); (ii) molecular weight; (iii) secondary and tertiary structure. Further biological assays are also essential to establish the relationship of a viroid with plant disease and to fulfill Koch's postulates.

Pest categorisation of 'Blight and blight-like' diseases of citrus.

The EFSA Panel on Plant Health performed a pest categorisation of 'Blight and blight-like' for the EU territory. Blight is a major disease of citrus. Similar 'blight-like' diseases are also known (e.g. declinio, declinamiento) and are addressed simultaneously with Blight in the present categorisation. The causal agent(s) remain(s) unknown and the potential role of a recently identified citrus endogenous pararetrovirus (Citrus Blight-associated pararetrovirus, CBaPRV) remains to be established. Transmissibility and ability to produce consistent (although poorly specific) symptoms have been demonstrated and a combination of indirect approaches is used, with limits, for diagnosis. There are large uncertainties on the biology of the causal agent(s) and on the epidemiology of the disease, including the transmission mechanism(s) responsible for the observed field spread. Blight has been reported from North, Central and South America, Africa and Oceania but is not known to occur in the EU. It is listed in Annex IIA of Directive 2000/29EC. It has the potential to enter, establish and spread in the EU territory. The main entry pathway (citrus plants for planting) is closed by existing legislation and entry is only possible on minor pathways (such as illegal import). Blight is a severe disease and a negative impact is expected should it be introduced in the EU, but the magnitude of this negative impact is very difficult to estimate. 'Blight and blight like' satisfies all criteria evaluated by EFSA to qualify as a Union quarantine pest. It does not meet the criterion of being present in the EU to qualify as a Union regulated non-quarantine pest (RNQP). Since the identity of the causal agent(s) of the Blight and blight-like disease(s) and the existence and efficiency of natural spread mechanism(s) remain unknown, large uncertainties affect all aspects of the present pest categorisation.

Pest categorisation of Citrus tristeza virus (non-European isolates).

The Panel on Plant Health performed a pest categorisation of non-European isolates of Citrus tristeza virus (CTV) for the EU territory. CTV is a well characterised virus for which efficient detection assays are available. It is transmitted by vegetative multiplication of infected hosts and by aphid vectors. The most efficient one, Toxoptera citricida, has limited EU presence but another one, Aphis gossypii, is broadly distributed. CTV is reported from a range of countries outside the EU and EU isolates are present in seven of the eight citrus-growing member states. Non-EU isolates are not known to occur in the EU and therefore do not meet one of the criteria for being a Union regulated non-quarantine pest. The natural host range of CTV is restricted to Citrus, Fortunella and Poncirus species. CTV non-EU isolates are listed in Annex IIAI of Directive 2000/29/EC and the main pathway for entry, plants for planting, is closed by the existing legislation. CTV isolates may therefore only enter through minor alternative pathways. They have the potential to subsequently spread through plants for planting and through the action of aphid vectors. CTV non-EU isolates are able to cause severe symptoms on a range of citrus crops that EU isolates do not induce. Overall, non-EU CTV isolates meet all the criteria evaluated by EFSA to qualify as Union quarantine pests. The main knowledge gaps and uncertainties concern (1) the status of Rutaceae species other than Citrus, Fortunella and Poncirus as natural hosts for CTV; (2) the potential undetected presence of non-EU CTV isolates in the EU and in particular the prevalence and biological properties of CTV isolates that may be present in ornamental citrus; and (3) the inability of EU CTV isolates apparently related to non-European stem pitting (SP) isolates to cause SP in sweet orange.

Pest categorisation of Satsuma dwarf virus.

The EFSA Panel on Plant Health performed a pest categorisation of Satsuma dwarf virus (SDV) for the EU territory. SDV is a well-known pathogen and the type species of the genus Sadwavirus in the family Secoviridae. SDV is now considered to include several other formerly distinct viruses which are therefore also covered in the present opinion. Citrus species and their relatives represent the main hosts of SDV and efficient diagnostic techniques are available. SDV is listed on some of its known hosts in Annex IIAI of Directive 2000/29/EC. It is transmitted by vegetative propagation of infected hosts and presumably through the soil, but the precise mechanism or vector(s) are still unknown. SDV is present in Asia and is not known to occur in the EU. Therefore, it does not meet this criterion to qualify as a Union regulated non-quarantine pest (RNPQ). Plants for planting represent the main pathway for the entry, but this pathway is closed by existing legislation for the main hosts (Citrus, Fortunella and Poncirus). SDV is, however, able to enter the EU on plants for plants of its unregulated rutaceous or non-rutaceous hosts. Should it be introduced, SDV has the potential to establish and subsequently spread with plants for planting and, possibly, through its poorly characterised natural spread mechanism(s). SDV is able to cause severe symptoms, quality and yield losses on a range of citrus crops. Overall, SDV meets all the criteria evaluated by EFSA to qualify as a Union quarantine pest. The main knowledge gaps and uncertainties concern (1) the potential significance of the unregulated rutaceous and non-rutaceous hosts for virus dissemination and epidemiology, (2) the origin and trade volume of the plants for planting of these host imported in the EU and (3) the efficiency of natural spread of SDV under EU conditions.

Pest categorisation of Tatter leaf virus.

The EFSA Panel on Plant Health performed a pest categorisation of Citrus tatter leaf virus (CTLV) for the EU territory. This virus is the causal agent of tatter leaf and graft incompatibility in trifoliate orange (Poncirus trifoliata) and its hybrids. CTLV is now recognised as a synonym of Apple stem grooving virus (ASGV), the type Capillovirus species, for which efficient diagnostics are available. There are no known ASGV vectors. The virus is reported in citrus from many countries. In the EU, while ASGV is widely present on apple and pear, it has never been reported on citrus. Since the citrus plants for planting pathway is closed by existing legislation, the main pathway for entry is plants for planting of other host species. In the EU, the high prevalence of ASGV in non-citrus hosts, but its absence in citrus ones suggests that interspecific host transfers are rare. However, there are high uncertainties on the importance and specifics of such host change events. No limits to the establishment of ASGV are identified and spread is likely through the vegetative propagation and trade of infected hosts. Infection of sensitive citrus rootstocks leads to stunted growth and decline of the entire plant a few years after grafting. The rootstocks that are now widely used to prevent citrus tristeza decline are the most affected. Among the criteria evaluated by EFSA for an organism to qualify as a Union quarantine pest, ASGV does not meet the criterion of being absent from or under official control in the EU territory. ASGV satisfies all the criteria evaluated by EFSA to qualify as a Union regulated non-quarantine pest. The main uncertainties concern the possible unreported presence of ASGV in citrus in the EU, the existence and efficiency of interspecific host transfers and the existence of ASGV natural spread.

Pest categorisation of naturally-spreading psorosis.

The EFSA Panel on Plant Health performed a pest categorisation of naturally-spreading psorosis of citrus for the European Union. Naturally-spreading psorosis is poorly defined, because the status of both the disease and its causal agent(s) is uncertain. However, Citrus psorosis virus (CPsV) is a well- characterised Ophiovirus that is systematically associated with the psorosis disease and therefore considered to be its causal agent. Efficient diagnostics are available for CPsV. It is present in at least three EU MS. Naturally-spreading psorosis is currently regulated by Directive 2000/29/EC, while CPsV is not explicitly mentioned in this Directive. CPsV has the potential to enter, establish and spread in the EU territory. However, the main pathway for entry is closed by the existing legislation so that entry is only possible through minor alternative pathways. Plants for planting are the major means of spread while there are uncertainties on the existence and efficiency of a natural spread mechanism. CPsV introduction and spread in the EU would have negative consequences on the EU citrus industry. Of the criteria evaluated by EFSA to qualify as a Union quarantine pest or as a Union regulated non-quarantine pest (RNQP), Naturally-spreading psorosis does not meet the criterion of being a well characterised pest or disease. As it is not explicitly mentioned in the legislation, it is unclear whether CPsV meets the criterion of being currently regulated or under official control. It meets, however, all the RNQP criteria. The key uncertainties of this categorisation concern: (1) the causal role of CPsV in the psorosis disease as well as elements of its biology and epidemiology, (2) the exact nature of the Naturally-spreading psorosis syndrome and the identity of its causal agent and, consequently, (3) whether CPsV should be considered as being covered by the current legislation.

Pest categorisation of Citrus leprosis viruses.

The EFSA Panel on Plant Health performed a pest categorisation of the Citrus leprosis viruses for the EU territory and identified five distinct viruses, Citrus leprosis virus C (CiLV-C), Citrus leprosis virus C2 (CiLV-C2), Hibiscus green spot virus 2 (HGSV-2), the Citrus strain of Orchid fleck virus (OFV) and Citrus leprosis virus N sensu novo (CiLV-N) as causing this severe disease, most significantly in sweet orange and mandarin. These viruses have in common that they do not cause systemic infections in their hosts and that they all are transmitted by Brevipalpus spp. mites (likely but not confirmed for HGSV-2). Mites represent the most important means of virus spread, while plants for planting of Citrus are only considered of minor significance. These well characterised viruses occur in South and Central America. Leprosis is currently regulated in directive 2000/29 EC and, together with its associated viruses, has never been recorded in the EU. All five viruses have the potential to enter into, establish in and spread within the EU territory, with plants for planting of non-regulated hosts, fruits of Citrus and hitch-hiking of viruliferous mites identified as the most significant pathways. Given the severity of the leprosis disease, the introduction and spread of the various viruses would have negative consequences on the EU citrus industry, the magnitude of which is difficult to evaluate given the uncertainties affecting the Brevipalpus spp. vectors (identity, distribution, density, transmission specificity and efficiency). Overall, leprosis and its five associated viruses meet all the criteria evaluated by EFSA to qualify as Union quarantine pests, but do not fulfil those of being present in the EU or of plants for planting being the main spread mechanism to qualify as Union regulated non-quarantine pests. The main uncertainties affecting this categorisation concern the Brevipalpus spp. mite vectors.

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.

Virus-viroid interactions: Citrus Tristeza Virus enhances the accumulation of Citrus Dwarfing Viroid in Mexican lime via virus-encoded silencing suppressors.

An assay to identify interactions between Citrus Dwarfing Viroid (CDVd) and Citrus Tristeza Virus (CTV) showed that viroid titer was enhanced by the coinfecting CTV in Mexican lime but not in etrog citron. Since CTV encodes three RNA silencing suppressors (RSSs), p23, p20 and p25, an assay using transgenic Mexican limes expressing each RSS revealed that p23 and, to a lesser extent, p25 recapitulated the effect observed with coinfections of CTV and CDVd.

Plant viroids: isolation, characterization/detection, and analysis.

When Diener discovered Potato spindle tuber viroid in 1971 (Diener, Virology 45:411-428, 1971), only a limited number of techniques were available for plant virus detection and purification. Biological assays using indicator hosts showing characteristic symptoms of infection and able to support high levels of viroid replication played a critical role in viroid detection and characterization. Polyacrylamide gel electrophoresis (PAGE) was the first molecular technique to be used for the rapid (2-3 days) identification of viroid-infected plants. Because it is the only diagnostic method that is sequence-independent, PAGE under denaturing conditions continues to play a key role in the identification of new viroids. Starting in the early 1980s, dot blot hybridization began to replace PAGE for routine viroid diagnosis. The first diagnostic protocols based on reverse transcription-polymerase chain reaction (RT-PCR) appeared approximately 10 years later, and much effort has subsequently been devoted to simplifying the sample preparation procedure and identifying group-specific primer pairs. This chapter describes four simple, easy-to-follow protocols-two involving PAGE and two others based on enzymatic amplification of viroid cDNAs-that currently play key roles in viroid discovery and characterization.

Microarray analysis of Etrog citron (Citrus medica L.) reveals changes in chloroplast, cell wall, peroxidase and symporter activities in response to viroid infection.

Viroids are small (246-401 nucleotides), single-stranded, circular RNA molecules that infect several crop plants and can cause diseases of economic importance. Citrus are the hosts in which the largest number of viroids have been identified. Citrus exocortis viroid (CEVd), the causal agent of citrus exocortis disease, induces considerable losses in citrus crops. Changes in the gene expression profile during the early (pre-symptomatic) and late (post-symptomatic) stages of Etrog citron infected with CEVd were investigated using a citrus cDNA microarray. MaSigPro analysis was performed and, on the basis of gene expression profiles as a function of the time after infection, the differentially expressed genes were classified into five clusters. FatiScan analysis revealed significant enrichment of functional categories for each cluster, indicating that viroid infection triggers important changes in chloroplast, cell wall, peroxidase and symporter activities.

The Mn-binding proteins of the photosystem II oxygen-evolving complex are decreased in date palms affected by brittle leaf disease.

Brittle leaf disease or maladie des feuilles cassantes (MFC) is a disorder affecting date palms (Phoenix dactylifera L.) which after a long declining process eventually leads to the death of the plant. No causal agent for the disease has been found so far but leaflets of affected palms are Mn-deficient despite the existence of adequate exchangeable Mn in the soils in which affected palms grow. The disease is specifically associated with an increase in a series of chloroplastic RNAs. A proteomic analysis of leaflets of affected and unaffected date palms showed differences in quantities of several proteins. Mn-binding PSBO and PSBP proteins, components of the oxygen-evolving complex of photosystem II, were decreased in affected tissue, reinforcing the relation between MFC and Mn deficiency. The quantities of other proteins were increased by disease suggesting a response to stress.

Two nucleotide positions in the Citrus exocortis viroid RNA associated with symptom expression in Etrog citron but not in experimental herbaceous hosts.

Citrus exocortis viroid (CEVd) is the causal agent of exocortis disease of citrus. CEVd has a wide host range that includes woody and herbaceous species. A new CEVd strain (CEVd(COL)), phylogenetically clustering with CEVd variants of Class A inducing severe symptoms in tomato, was identified in Colombia and shown to induce only extremely mild symptoms in Etrog citron indicator plants. Using site-directed mutagenesis, two nucleotide substitutions (314A → G and 315U → A) in the lower strand of the P domain of the predicted CEVd(COL) secondary structure resulted in a severe artificial CEVd(MCOL) variant. Conversely, two nucleotide exchanges (314G → A and 315A → U) in the same region of the severe variant CEVd(E-117) resulted in a symptomless artificial CEVd(ME-117) variant. Infectivity assays conducted with the natural and mutated variants showed that all induced severe symptoms in Gynura aurantiaca, tomato and chrysanthemum. This is the first report of the identification of pathogenic determinants of CEVd in citrus, and shows that these pathogenicity determinants are host dependent.

Molecular and biological characterization of natural variants of Citrus dwarfing viroid.

Citrus dwarfing viroid has been proposed as an agent to control tree size in high-density plantations. Thirty-three field isolates have been characterized, and the most frequent sequence/s have been identified. Five distinct variants were selected for biological characterization. Symptom expression analysis demonstrated a good correlation between leaf/stem symptoms and plant growth. The discriminating nucleotide sequence differences included two deletions and an insertion resulting in a reorganization of the base pairing of the terminal left loop, two (G42 --> A and C52 --> U) changes found in one of the variants, and as many as thirteen changes located in the right and left regions flanking the CCR.

An artificial chimeric derivative of Citrus viroid V involves the terminal left domain in pathogenicity.

The recently described Citrus viroid V (CVd-V) induces, in Etrog citron, mild stunting and very small necrotic lesions and cracks, sometimes filled with gum. As Etrog citron plants co-infected with Citrus dwarfing viroid (CDVd) and CVd-V show synergistic interactions, these host-viroid combinations provide a convenient model to identify the pathogenicity determinant(s). The biological effects of replacing limited portions of the rod-like structure of CVd-V with the corresponding portions of CDVd are reported. Chimeric constructs were synthesized using a novel polymerase chain reaction-based approach, much more flexible than those based on restriction enzymes used in previous studies. Of the seven chimeras (Ch) tested, only one (Ch5) proved to be infectious. Plants infected with Ch5 showed no symptoms and, although this novel chimera was able to replicate to relatively high titres in singly infected plants, it was rapidly displaced by either CVd-V or CDVd in doubly infected plants. The results demonstrate that direct interaction(s) between structural elements in the viroid RNA (in this case, the terminal left domain) and as yet unidentified host factors play an important role in modulating viroid pathogenicity. This is the first pathogenic determinant mapped in species of the genus Apscaviroid.

Effect of citrus hosts on the generation, maintenance and evolutionary fate of genetic variability of citrus exocortis viroid.

Citrus exocortis viroid (CEVd) populations are composed of closely related haplotypes whose frequencies in the population result from the equilibrium between mutation, selection and genetic drift. The genetic diversity of CEVd populations infecting different citrus hosts was studied by comparing populations recovered from infected trifoliate orange and sour orange seedling trees after 10 years of evolution, with the ancestral population maintained for the same period in the original host, Etrog citron. Furthermore, populations isolated from these trifoliate orange and sour orange trees were transmitted back to Etrog citron plants and the evolution of their mutant spectra was studied. The results indicate that (i) the amount and composition of the within-plant genetic diversity generated varies between these two hosts and is markedly different from that which is characteristic of the original Etrog citron host and (ii) the genetic diversity found after transmitting back to Etrog citron is indistinguishable from that which is characteristic of the ancestral Etrog citron population, regardless of the citrus plant from which the evolved populations were isolated. The relationship between the CEVd populations from Etrog citron and trifoliate orange, both sensitive hosts, and those from sour orange, which is a tolerant host, is discussed.

Transcriptional response of Citrus aurantifolia to infection by Citrus tristeza virus.

Changes in gene expression of Mexican lime plants in response to infection with a severe (T305) or a mild (T385) isolate of Citrus tristeza virus (CTV) were analyzed using a cDNA microarray containing 12,672 probes to 6875 different citrus genes. Statistically significant (P<0.01) expression changes of 334 genes were detected in response to infection with isolate T305, whereas infection with T385 induced no significant change. Induced genes included 145 without significant similarity with known sequences and 189 that were classified in seven functional categories. Genes related with response to stress and defense were the main category and included 28% of the genes induced. Selected transcription changes detected by microarray analysis were confirmed by quantitative real-time RT-PCR. Changes detected in the transcriptome upon infecting lime with T305 may be associated either with symptom expression, with a strain-specific defense mechanism, or with a general response to stress.

Host Effect on the Molecular and Biological Properties of a Citrus exocortis viroid Isolate from Vicia faba.

ABSTRACT Citrus exocortis viroid (CEVd) is the casual agent of citrus exocortis disease, and has been found in naturally infected citrus and noncitrus hosts. Field isolates of CEVd may infect susceptible hosts as a complex of genetically related sequence variants (haplotypes). In the present work, a CEVd isolate recovered from a symptomless broad bean plant was characterized as a heterogeneous population with a nucleotide diversity of 0.026, which did not contain a predominant haplotype. When nucleic acid extracts of this infected broad bean were used to inoculate tomato, the plants displayed symptoms and the CEVd population was more homogeneous, with a nucleotide diversity of 0.007. However, when nucleic acid extracts from this tomato isolate were back inoculated to new broad bean plants, this isolate did not revert to the original population, because it showed low nucleotide diversity (0.001) and induced symptoms in the broad bean plants. Symptomless broad bean plants may act as reservoirs of highly heterogeneous populations of CEVd variants, providing an excellent inoculum source in terms of its potential to infect a broad range of putative hosts. The epidemiological implications are discussed.