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Chrysanthemum stunt pospiviroid

2002; Wiley; Volume: 32; Issue: 2 Linguagem: Inglês

10.1046/j.1365-2338.2002.00583.x

1365-2338

Plant and Fungal Interactions Research

EPPO BulletinVolume 32, Issue 2 p. 245-253 Free Access Chrysanthemum stunt pospiviroid First published: 05 September 2002 https://doi.org/10.1046/j.1365-2338.2002.00583.xCitations: 4 Roche Diagnostics is the new trading name for Boehringer Mannheim, the name normally associated with the DIG-labelling probe system. Roche Diagnostics est le nouveau nom commercial de Boehringer Mannheim, le nom normalement associé au système de sondes marquées à la digoxygénine. AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Specific scope This standard describes a diagnostic protocol for Chrysanthemum stunt pospiviroid. Champ d’application spécifique Cette norme décrit un protocole de diagnostic pour le Chrysanthemum stunt pospiviroid. Specific approval and amendment First approved in 2001-09. Approbation et amendement spécifiques Approbation initiale en 2001-09. Introduction Chrysanthemum stunt is an economically important disease of florists’ chrysanthemum (Dendranthema × grandiflorum) and especially all-year-round cultivars. Found in most (if not all) chrysanthemum-growing regions of the world, the causal agent of the disease is Chrysanthemum stunt viroid pospiviroid (CSVd), a close-relative of Potato spindle tuber pospiviroid. CSVd contains no proteins but consists solely of a single-stranded, circular RNA of between 354 and 356 nucleotides in length. However, due to internal sequence complementarity, the viroid RNA folds and binds internally to form a double-stranded molecule, which exhibits a high degree of secondary structure and makes CSVd exceptionally stable. As a result of this, spread can occur mechanically (especially by contact with contaminated tools or plant-to-plant contact), although propagation from infected mother plants is the major means of CSVd transmission. Evidence for the transmission of CSVd by seed, dodder or insect vectors is not strong but, if transmission does occur by any of these means, it is rare. For more general information about CSVd, see EPPO/CABI (1997). Introduction Le rabougrissement du chrysanthème est une maladie d’importance économique du chrysanthème des fleuristes (Dendranthema× grandiflorum), et particulièrement des cultivars qui produisent toute l’année. L’agent causal de la maladie est présent dans la plupart des régions productrices de chrysanthèmes du monde (si ce n’est toutes). Il s’agit du Chrysanthemum stunt pospiviroid (CSVd), étroitement apparenté au Potato spindle tuber viroid (PSTVd). Le CSVd ne contient pas de protéines, il est constitué simplement d’un ARN circulaire monocaténaire mesurant entre 354 et 356 nucléotides de longueur. Cependant, en raison d’une complémentarité de séquences interne, l’ARN du viroïde se replie et se lie à lui-même pour former une molécule bicaténaire, qui présente une forte structure secondaire et rend le CSVd exceptionnellement stable. En conséquence, la dissémination peut être mécanique (en particulier par contact avec des outils contaminés ou entre les plantes), même si la propagation à partir de plantes mères infectées constitue le principal moyen de transmission du CSVd. Il est peu probable que le CSVd soit transmis par les semences, les cuscutes ou des insectes vecteurs, mais si c’est le cas, cela reste rare. Pour plus d’informations générales sur le CSVd, voir EPPO/CABI (1997). Identity Name: Chrysanthemum stunt viroid Synonyms: Chrysanthemum stunt mottle virus (in part) American stunt virus (in part) Taxonomic position: Family Pospiviroidae, Genus Pospiviroid Acronym: CSVd Bayer computer code: CSVD00 Phytosanitary categorization: EPPO A2 list no. 92; EU Annex designation II/A2 Identité Nom: Chrysanthemum stunt viroid Synonymes: Chrysanthemum stunt mottle virus (en partie), American stunt virus (en partie) Classement taxonomique: Famille Pospiviroidae, genre Pospiviroid Acronyme: CSVd Code informatique Bayer: CHSXXX Catégorisation phytosanitaire: Liste A2 de l’OEPP no. 92; Désignation Annexe UE II/A2 Detection Hosts The only important host of CSVd is florists’ chrysanthemum (Dendranthema × grandiflorum). CSVd has also been reported as a natural infection on Argyranthemum frutescens, Petunia hybrida Surfinia hybrids and Ageratum spp., but all are considered to be minor hosts. In addition, a range of other hosts from the Asteraceae, as well as tomato (Lycopersicon esculentum), can be infected experimentally by sap inoculation or grafting. Disease symptoms In many chrysanthemum cultivars, up to 30% of infected plants are symptomless. When symptoms are seen, they are often variable and are highly dependent on environmental conditions, especially temperature and light. The main symptom is, as the name of the organism implies, stunting, with a reduction of up to 50% in overall height in mature plants. Stems also become very brittle, readily breaking at the branch point. The other common symptoms are floral, with infected plants having reduced flower size and demonstrating premature flowering, sometimes by up to 10 days. Less commonly, flowering can also be delayed. In certain cultivars, especially red-pigmented ones, symptoms can also include flower break or bleaching (a reduction in colour intensity). Foliar symptoms are less common and the presence of pale, upright young leaves is often the only indication of infection. Leaf spots or flecks, often associated with leaf distortions (crinkling), are also sometimes seen, the most extreme example being the symptom described as ‘measles’, which appears as large, yellow leaf blotches. However, this symptom is restricted to a handful of cultivars (e.g. cvs. Mistletoe and Bonnie Jean) and, as most of these cultivars are no longer commercially available, is very rarely seen in naturally infected plants. The symptoms of CSVd on Argyranthemum frutescens are similar to those for Dendranthema, with stunting and premature flowering observed in some cultivars. On surfinias, there are generally no symptoms, although stunting is seen with some cultivars (W. Menzel, University of Hannover, DE, pers. comm.). Sampling Because of the high incidence of symptomless carriers, at least 10% of the total should be examined by visual inspection. Due to variability in symptoms, laboratory-based tests should be used to confirm suspect plants. With regard to the timing of sampling, some early research indicated that CSVd concentrations within infected plants decrease during the winter months (November to February in the Northern Hemisphere) and it is suggested that sampling during these months is best avoided. However, using more modern, sensitive tests, it has proved possible to detect CSVd at all times of the year, indicating that while spring and summer sampling is preferable, it is not critical. The amount and type of tissue sampled depends on the test performed but, for the molecular methods described here, even a single leaf should be sufficient. CSVd distribution appears to be uniform within infected mature plants, but it is nevertheless recommended to take several fully grown leaves from a number of different stems of the same plant. Stems can also be tested, but are harder to sample and process. Détection Hôtes Le chrysanthème des fleuristes (Dendranthema × grandiflorum) est le seul hôte important du CSVd. Il a également été signalé infectant naturellement Argyranthemum frutescens, Petunia hybrida hybrides Surfinia et Ageratum spp., mais tous sont considérés comme des hôtes mineurs. En outre, une gamme de plantes-hôtes de la famille des Asteraceae, ainsi que la tomate (Lycopersicon esculentum), peuvent être infectées expérimentalement par inoculation de sève ou greffe. Symptômes de maladie Dans de nombreux cultivars de chrysanthème, jusqu’à 30% des plantes infectées ne présentent pas de symptômes. Lorsque des symptômes sont observés, ils sont souvent variables et dépendent fortement des conditions environnementales, en particulier de la température et de la lumière. Le principal symptôme est un rabougrissement, comme le nom de l’organisme l’indique, avec une réduction de la taille totale des plantes matures pouvant atteindre 50%. Les tiges sont également très cassantes et se rompent facilement aux nœuds. Les autres symptômes courants sont observés sur les fleurs. Leur taille est réduite et la floraison est précoce, parfois anticipée de 10 jours. Dans des cas plus rares, la floraison est retardée. Pour certains cultivars, en particulier ceux à floraison rouge, les symptômes peuvent inclure également une panachure ou une décoloration de l’inflorescence (réduction de l’intensité des couleurs). Les symptômes foliaires sont moins courants et la présence de jeunes feuilles pâles et dressées est souvent la seule indication de l’infection. Une moucheture ou tacheture des feuilles, souvent associées à des déformations (‘frisolée’), sont également parfois observées; l’exemple le plus extrême étant le symptôme décrit sous le nom de ‘rougeole’ qui apparaît comme une marbrure diffuse jaune. Toutefois, ce symptôme ne concerne que quelques cultivars (par exemple les cvs. Mistletoe et Bonnie Jean) et il est rarement observé dans des plantes infectées naturellement étant donné que la plupart de ces cultivars ne sont plus disponibles commercialement. Le CSVd produit des symptômes similaires sur Argyranthemum frutescens, avec un rabougrissement et une floraison prématurée chez certains cultivars. Les surfinias ne présentent généralement pas de symptômes, même si un rabougrissement est observé chez certains cultivars (W. Menzel, université de Hanovre, comm. pers.). Echantillonnage En raison de la forte incidence des porteurs asymptômatiques, au moins 10% du total d’une culture entière doit être examiné par inspection visuelle. Compte tenu de la diversité des symptômes, des tests de laboratoire doivent être réalisés pour confirmer tout symptôme suspect. En ce qui concerne les périodes d’échantillonnage, des résultats anciens ont montré que les concentrations du CSVd dans les plantes infectées décroissent pendant les mois d’hiver (novembre à février dans l’Hémisphère nord) et ils suggéraient d’éviter d’échantillonner pendant cette période. Cependant, des tests plus sensibles et modernes permettent de détecter le CSVd à tout moment de l’année. Il est toujours préférable de prélever les échantillons au printemps ou en été, mais cela n’a plus une importance critique. La quantité et le type des tissus échantillonnés dépendent du test employé. Une seule feuille devrait en principe suffire pour les méthodes moléculaires décrites ici. Le CSVd a une répartition apparemment uniforme dans les plantes matures infectées, mais il est néanmoins recommandé de prélever plusieurs feuilles complètement développées sur un certain nombre de tiges d’une même plante. Les tiges peuvent également être testées, mais l’échantillonnage et le test sont plus difficiles. Identification For identifying CSVd in samples, four methods are available: • return polyacrylamide gel electrophoresis (R-PAGE) – this only permits viroid detection and not identification [the latter only when R-PAGE is combined with nucleic acid hybridization (Northern blot) analysis]; • nucleic acid hybridization using a digoxigenin (DIG)-labelled cRNA probe – this is sensitive and reliable but only economical when large numbers of samples are to be analysed; • reverse transcriptase-polymerase chain reaction (RT-PCR) – this is the preferred method of detection and identification when combined with restriction fragment length polymorphism (RFLP) analysis of RT-PCR products and when equipment is unavailable for TaqMan (see below); • fluorogenic 5′-nuclease assay (TaqMan) – this is a very sensitive and specific method for CSVd detection and identification. It is the preferred method if equipment is available. R-PAGE method This method was developed by J.W. Roenhorst (Roenhorst et al., 2000). It is an improved version of the protocol given in EPPO Phytosanitary Procedure No. 24 (OEPP/EPPO, 1989). Sample preparation Sap is extracted from several leaves (up to 1 g) using a leaf press, e.g. Pollähne Press (Meku, Germany) and 0.5 mL added to a 2-mL centrifuge tube, containing 250 µL of extraction buffer (1 m Tris-HCl pH 8.0, 100 mm NaCl, 10 mm EDTA, 2% (w/v) SDS) and 750 µL of water-saturated phenol (containing 0.1% 8-hydroxyquinoline). After adding the sap, the contents of the tube are mixed by vortexing for 90 s and then centrifuged at 12 000 g for 10 min at 4 °C. A portion of the upper, aqueous phase (400 µL) is then transferred to another centrifuge tube containing 1 mL of 96% ethanol and 40 µL of 3 m sodium acetate, pH 5.6. The RNA is then precipitated by incubation at −20 °C for at least 1 h, before centrifugation as described before. The supernatant is then removed and the pellet dried, before being resuspended in 30 µL of RNase-free water, containing 0.05% xylene cyanol FF (added as an electrophoresis dye marker). Samples can be stored at −20 °C for at least one week. Before loading, samples are centrifuged for 2 min at 12 000 g to remove any insoluble material. Gel preparation Polyacrylamide gels are prepared on GelBond PAGfilm (124 × 258 mm) using a Multiphor II casting kit (Amersham Pharmacia Biotech). Before use, the glass plates are coated with Repel-Silane ES (Amersham Pharmacia Biotech). Gels are prepared to a thickness of 1 mm, using a 5% acrylamide mix: 3.75 mL of 40% (w/v) acrylamide/bis solution (ratio 37.5:1; Bio-Rad), 1.5 mL of 10 × TBE stock (890 mm Tris pH 8.3, 890 mm boric acid, 20 mm EDTA; Bio-Rad), 36 µL of TEMED and 200 µL of 10% ammonium persulfate (freshly prepared), made up to a total volume of 30 mL with distilled water. The wells are formed using a 40-tooth comb (giving wells of 4 × 4 × 0.25 mm). Gel electrophoresis Electrophoresis is performed using the horizontal Multiphor II system (Amersham Pharmacia Biotech), in combination with a power supply (capable of delivering up to 400 V) and a circulating chiller-heater unit (e.g. Amersham Pharmacia Biotech MultiTemp III). Once the gel has been prepared, it is placed onto the ceramic plate of the Multiphor unit, which has been precoated with a thin layer of melting-point bath oil (Sigma, Cat. No. M-6884) and aligned so that the leading edge of the wells is at position 4. Paper wicks (250 × 50 mm × 1.2 mm thick; Schleicher & Schull, GB004), which have been soaked in 1.67 × TBE, are then arranged to overlay the gel edges by 10–15 mm. The samples (5 µL) are then loaded into the wells. At least one positive control and one negative control is included. A glass plate (125 × 260 × 3 mm) is then placed on the inner edges of the wicks, thereby covering the whole surface of the gel. The apparatus cover is then replaced, so that the electrodes make contact about 5 mm in from the outer edge of the wicks. The first run is performed under native conditions at 15 °C and a constant current of 20 mA. After about 6 min, the current is increased to 30 mA and the run continued for approximately another 55 min, by which time the dye marker has reached marker 8 on the ceramic plate. At this point, the valve controlling the flow from the water bath to the ceramic plate is closed, and the temperature of the water bath is then adjusted to 75 °C. While this heats, the run is continued for another 15 min, or until the dye front is between positions 10 and 11 and the water temperature of the disconnected water bath has reached 75 °C. Prior to the return run (under denaturing conditions), the paper wicks are replaced with fresh ones and the gel and wicks covered by two polyester sheets, to prevent dehydration. The first sheet (95 × 270 mm) is placed between the paper wicks (covering the gel) and the second (175 × 270 mm) covers both the gel and the wicks. The glass plate and electrodes are then replaced and the valve carefully opened to achieve a gradual heating of the gel. The temperature of the water bath is then reduced to 60 °C, the polarity reversed and the return run started; initially at a constant 2 mA for 8 min, before increasing the current to 50 mA and running for 25–30 min, by which time the loading dye marker should have reached the left-hand wick. The gel is then removed from the ceramic plate and carefully rinsed in 96% ethanol to remove any residual oil, before rinsing in distilled water. Silver staining After electrophoresis is finished, the gel is fixed for 10 min in 10% ethanol containing 0.5% acetic acid (made up in distilled water). Fixing and all subsequent stages are done at room temperature with gentle shaking. The gel is transferred into 10 mm silver nitrate for 20 min, before washing twice in distilled water (1 min each). The gel is then transferred into a solution of 375 mm sodium hydroxide containing 2.3 mm sodium borohydride and 0.4% formaldehyde (37% w/v) and left for 3–5 min, to allow the stain to develop, before stopping for 10 min in 1% acetic acid. In positive samples, the viroid band should appear as a distinct band, separated from the other nucleic acids. To store gels, incubate in 10% acetic acid and 5% glycerol for at least 15 min, before covering with cellophane, placing on a glass plate and leaving to dry for around 2 days at room temperature. Nucleic acid hybridization method Probe preparation In the method described here, the clone used is pCSVD-5 (generated by R.A. Mumford, Central Science Laboratory, GB), which consists of a single, full-length cDNA copy of CSVd, cloned into a pGEM plasmid vector (Promega). Other clones, such as pCS9 (Candresse et al., 1990), are very similar constructs and could also be used. Plasmid is purified using a Wizard kit (Promega) or similar, following the manufacturer’s instructions. Purified plasmid is cut using restriction enzyme Sma I (Promega), following the conditions recommended by the manufacturer. The linearized plasmid is purified by phenol:chloroform extraction and ethanol precipitation, using standard methods. After precipitation, the plasmid pellet is washed in 70% ethanol, dried and resuspended in 50 µL of sterile distilled water. The plasmid is then quantified using a Nucleic dot metric quantitation kit (VH Bio, UK) or a similarly accurate method. DIG-labelled probe is synthesized by in-vitro transcription. Reactions are performed by adding 1 µg of linearized plasmid to 1 × transcription buffer (Promega), 10 mm DTT, 1 × DIG RNA labelling mix (Roche Diagnostics1), 40 U RNasin (Promega) and 20 U of T7 RNA polymerase (Promega). The reaction is made up to a total volume of 20 µL, using RNase-free water and incubated at 37 °C for up to 2 h. Following synthesis, the amount of labelled probe produced is quantified against a known DIG-labelled RNA standard (Roche Diagnostics), using the recommended method. cRNA probes should be stored at −20 °C (or below), where they will remain stable for up to a year. Sample and test membrane preparation Samples are prepared using a method adapted from Podleckis et al. (1993). Leaf tissue (up to 0.5 g) is ground in AMES extraction buffer (3% SDS, 20% ethanol, 1 m NaCl, 0.5 m sodium acetate, 10 mm MgCl2, pH 6.0), at a ratio of 1:1.5 (w/v). The resulting extract is then transferred to a 1.5-mL centrifuge tube and incubated at 37 °C for 15 min. Following incubation, an equal volume of chloroform is added, mixed until an emulsion has formed and centrifuged at maximum speed for 5 min, to separate the phases. The samples are then used immediately or can be stored at 4° until the next day (at most). For each sample, 3–5 µL of the aqueous upper phase is taken and pipetted directly onto the surface of a positively charged nylon membrane (Roche Diagnostics). In addition to the test samples, appropriate positive (infected and plasmid) and negative (uninfected and buffer) controls are also applied. After sample application, the membrane is dried (at 80 °C for 10 min), before fixation in a UV-crosslinker at 70 000 µJ cm−2; baking at 80 °C for 2 h can also be used for drying/fixing but background signals are increased. If required, at this stage, the membrane can be stored for several days in the dark at room temperature. Hybridization Hybridization is performed using the general conditions recommended by Roche Diagnostics, for use with DIG-labelled RNA probes. The test membrane is placed in a hybridization tube, containing 10 mL of DIG Easy-Hyb buffer (Roche Diagnostics) and prehybridized at 68 °C, in a hybridization oven (Hybaid). After 1–2 h, the prehybridization buffer is poured off and replaced with 6 mL of preheated DIG Easy-Hyb containing 100–250 ng of probe; the tube is returned to the oven and incubated overnight at 68 °C. Following hybridization, the membrane is transferred to a sandwich box and washed four times on an orbital/rocking platform shaker; two washes are performed at room temperature in 2 × SSC + 0.1% SDS for 30 min, followed by two further 15 min washes at 68 °C in 0.5 × SSC + 0.1% SDS. Detection Detection is performed using CSPD substrate (Tropix), following the protocol recommended by Roche Diagnostics for chemiluminescent detection of DIG-labelled RNA probes. When using Kodak X-Omat film, an exposure of 1 h is generally ideal for giving a clear result. However, a second exposure (longer or shorter) may be necessary depending on the strength of signal detected. Alternative method As an alternative for laboratories which are not equipped for performing hybridization tests, a commercially available CSVd hybridization test kit is available from Agdia (Elkhart, Indiana, US). With this system, the user prepares the samples and test membrane, which is then returned to Agdia for testing, who in turn supply the final results. This system has been shown to give identical results to the hybridization system described here. RT-PCR method This follows the method described in Mumford et al. (2000). Total nucleic acid extraction Total nucleic acid (TNA) is extracted from samples, using a method adapted from Lohdi et al. (1994). Leaf tissue (100 mg) is placed in a 10 × 15 cm 500-gauge polythene bag and frozen in liquid nitrogen, before being ground into a fine powder using a small hand roller. Grinding is continued until thawing begins and the tissue forms a smooth paste. One mL (10 volumes) of grinding buffer (2% CTAB, 100 mm Tris-HCl (pH 8.0), 20 mm EDTA, 1.4 m NaCl, 1.0% Na sulphite, 2.0% PVP-40) is added and mixed thoroughly using the roller. The ground sap is decanted into a 1.5-mL centrifuge tube and incubated at 65 °C for 10–15 min After incubation, the tubes are centrifuged at maximum speed for 5 min (at room temperature). Clarified sap (700 µL) is removed and placed in another microtube to which is added an equal volume of chloroform:iso-amyl alcohol (24:1 v:v) and mixed to emulsion by inverting the tube. It is centrifuged at maximum speed in a microfuge for 5 min (at room temperature). The upper (aqueous) layer is carefully removed and transferred to a fresh tube. An equal volume of chloroform:isoamyl alcohol added, mixed and spun as before. The aqueous layer is removed, taking extra care not to disturb the interphase, and 0.5 volumes of 5 m NaCl and an equal volume of ice-cold iso-propanol is added to it. It is well mixed and incubated at −20 °C overnight. The TNA is pelleted by centrifuging for 10 min at maximum speed. The salt/ethanol is decanted off and pellet is washed by adding 500 µL 70% ethanol and spinning for 3–4 min. The ethanol is decanted off and the pellet is air-dried to remove residual ethanol. The pellet is then re-suspended in 100 µL of molecular-biology grade water. RT-PCR One µL of TNA or control (infected RNA, uninfected RNA and water) is aliquoted into a 0.2-mL PCR tube and 20 pm of each primer Vir 1 (5′-CTTCAGTTGTTTCCACCGGGTAG-3′) and Vir 2 (5′-TTCCTGTGGTGCACTCCTGACC-3′) is added. A final volume of 15 µL is produced by adding molecular-biology grade water. The viroid RNA is denatured by heating to 95 °C for 3 min, before cooling to 4 °C (or below) for 5–10 min (for convenience, this can be done on a thermal cycler, if it has a cooling facility). Once the denaturation step has been completed, the remaining components of the reaction mix can be added (1.25 U Taq polymerase, 10 U AMV reverse transcriptase, 1 × Taq buffer containing 1.5 mM MgCl2 and 300 µM dNTPs; all components from Promega), in a total volume of 35 µL, made up with sterile, nuclease-free water. After mixing, the tubes are returned to the thermal cycler and RT-PCR is performed using the following program: 50 °C for 30 min, 95 °C for 4 min, followed by 30 cycles of 95 °C, 60 °C, 72 °C (each for 1 min), followed by a final extended extension soak of 72 °C for 5 min. Agarose gel electrophoresis Following RT-PCR, products are analysed by gel electrophoresis. PCR mix (12.5 µL) is combined with 2.5 µL of 6 × loading buffer and loaded onto a 2% agarose-TBE gel and run in 1 × TBE buffer, at 5–10 V cm−1. For sizing, a 100-bp ladder (AB Gene, UK) is also run on the gel, in addition to the samples. After running, the gel is stained in ethidium bromide (1 µg mL−1 in deionized water), before being visualized under UV light. Positive samples will give a band of 262 base pairs (bp). An image (film or digital) of the gel is captured to include with the final report. RFLP RT-PCR products from positive samples are purified using a PCR product purification kit (MN Nucleo-Spin Extract or similar). The purified product is then digested using Hind III restriction enzyme (Promega), in a reaction containing 17 µL of product, 10 U of enzyme, 2 µL of 10 × buffer. Reactions are then incubated for 2 h at 37 °C. Following incubation, the reaction products are then analysed by agarose gel electrophoresis, as described above. For products generated using the Vir primer pair, Hind III will cut CSVd products (to give a 220-bp band) but will not cut PSTVd. Fluorogenic 5′-nuclease assay (TaqMan) method This follows the method described in Mumford et al. (2000). Total nucleic acid extraction TNA is extracted using the same method as described above, for RT-PCR. TaqMan assay Reactions are set up, in duplicate, in 96-well reaction plates using reagents supplied by Applied Biosystems (unless otherwise stated). For each reaction, 1 µL of TNA extract is added to 7.5 pmol of reverse primer (CSVd 297R: 5′-GGAAAAAAAGGCGTTGAAGCTT-3′) and made up to a final volume of 5 µL, with molecular-biology grade water. This mix is then heated to 95 °C for 3 min before chilling on ice. After chilling for 5–10 min, the denatured RNA-reverse primer mix is then added to the remainder of the master mix containing 1 × Buffer A (supplied with AmpliTaq Gold), 5.5 mM MgCl2, 0.5 mM dNTPs, 7.5 pmol forward primer (CSVd 220F: 5′-CTGCCCTAGCCCGGTCTT-3′), 2.5 pmol of TaqMan probe (CSVd 249T: 5′-[FAM] -CAGTTGTTTCCACCGGGTAGTAGCCAA-[TAMRA]-3′), 0.625 U AmpliTaq Gold and 10 U of M-MLV RT (Promega), made up to a final volume of 25 µL, with molecular-biology grade water. Plates are then cycled at generic system conditions (48 °C for 30 min, 95 °C for 10 min and 40 cycles of 60 °C for 1 min, 95 °C for 15 s) within the 7700 Sequence Detection System (Applied Biosystems), using real-time data collection. After cycling, data is analysed using the Sequence Detector software package, supplied by Applied Biosystems. Identification Quatre méthodes sont disponibles pour identifier le CSVd dans les échantillons: • la R-PAGE électrophorèse inverse sur gel de polyacrylamide) – elle permet seulement de détecter des viroïdes, mais pas de les identifier; l’identification est possible seulement en combinant la R-PAGE avec l’hybridation moléculaire (Northern blot); • l’hybridation moléculaire avec une sonde d’ARNc marquée à la digoxygénine – elle est sensible et fiable mais économique seulement pour l’analyse de nombreux échantillons; • la RT-PCR (amplification génique après transcription réverse) – la méthode préférée de détection et d’identification lorsqu’elle est combinée avec l’analyse RFLP (polymorphisme de longueur des fragments de restriction) des produits de RT-PCR et lorsqu’on ne dispose pas du matériel nécessaire à la méthode TaqMan (voir ci-dessous); • la technique TaqMan – méthode très sensible et spécifique pour la détection et l’identification du CSVd. C’est la méthode préférée si la matériel est disponible. Méthode R-PAGE C’est la méthode développée par J.W. Roenhorst (Roenhorst et al., 2000). Il s’agit d’une version améliorée du protocole donné dans la Méthode phytosanitaire no. 24 de l’OEPP (OEPP/EPPO, 1989). Préparation des échantillons Le jus est extrait de plusieurs feuilles (jusqu’à 1 g) à l’aide d’une presse, par ex. du type presse Pollähne (Meku, DE). 0,5 mL sont ajoutés à un microtube de 2 mL, contenant 250 µL de tampon d’extraction (Tris-HCl 1 mà pH = 8,0, 100 mm de NaCl, 10 mm d’EDTA (acide éthylène diamine tétracétique disodique), SDS 2% (p/v)) et 750 µL de phénol saturé en eau (contenant 0,1% de 8-hydroxyquinoline). Après ajout du jus, le contenu du tube est mélangéà l’aide d’un agitateur type Vortex pendant 90 s, puis centrifugéà 12 000 g pendant 10 min à 4 °C. Une portion de la phase supérieure aqueuse (400 µL) est alors transférée à un autre microtube contenant 1 mL d’éthanol à 96% et 40 µL d’acétate de sodium 3 mà pH = 5,6. L’ARN est ensuite précipité par incubation à−20 °C pendant au moins 1 h, avant centrifugation comme décrit ci-dessus. Le surnageant est ens