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International audience

A highly sensitive assay, based on polymerase chain reaction amplification of cDNA synthesized from the viral RNA of antibody-captured viral particles, has been developed for plum pox potyvirus (PPV) detection. The reaction, called immunocapture/PCR (IC/PCR), yields a specific 243-bp product. The immunocapture step, by allowing the use of large sample volumes and by the viral particle prepurification it achieves, dramatically increases the sensitivity of the assay. As few as 8000 target viral particles per ml of plant extract could be detected by IC/PCR. When compared to direct PCR (Wetzel et al., 1991), molecular hybridization using 32P-labeled cRNA probes and ELISA, this result corresponds to a 250-fold, 625-fold and 5000-fold increased sensitivity, respectively. The high sensitivity of IC/PCR was confirmed during an indexing trial with field samples collected from naturally infected trees. This very powerful technique should have wide ranging applications for the detection of a number of other viruses and pathogens for which specific antisera and sequence data are available.

International audience; Analysis of the P1C124 source (associated with the cherry necrotic mottle leaf (CNML) disease) revealed the presence of two different viral agents. The complete nucleotide sequence of one of these agents, P1A, had an overall nucleotide sequence similarity of 83% with a previously sequenced North American isolate of Cherry green ring mottle virus (CGRMV) and should therefore be regarded as an European isolate of CGRMV. Approximately 2 kb of the 5′ end of the genome of the second agent, P1B, were also sequenced and were shown to be 82% homologous with Cherry necrotic rusty mottle virus (CNRMV), another member of the Foveavirus genus. The possible involvement of CGRMV-P1A and of CNRMV-P1B in the etiology of the CNML disease is discussed.

International audience

We have developed an immunocapture-PCR (IC-PCR) detection technique for plum pox potyvirus (PPV) which is both simple and highly sensitive. This single-day assay can detect about 2000 virus particles (200 fg of virus) diluted in 100 μl of crude plant sap, which is equivalent to a sensitivity about 2000 times better than that of a standard ELISA assay. RFLP analysis and sequencing of the amplified cDNA fragment indicate that three groups of strains with limited intra-group variability can be discriminated. Two of these groups correspond to the previously described D and M serotypes of PPV. The third group contains, so far, only the El Amar Egyptian isolate. Strains belonging to the D or M serotypes can easily be discriminated by Rsal polymorphism in the amplified cDNA fragment. Synthetic oligonucleotides allowing specific amplification of PPV strains belonging either to the D or to the M serotypes have also be designed.

International audience

International audience; Replacing nick-translated DNA probes by in vitro transcribed complementary RNA (cRNA) probes considerably increased the sensitivity of dot-blot detection tests of potato spindle tuber viroid and chrysanthemum stunt viroid. As compared to the limit of detection of 5–10 pg of viroid obtained with 32P-labelled DNA probes, cRNA probes allow the detection of less than 1 pg of pure viroid. When labelled with biotin by incorporation of biotin-labelled ribonucleotides, the cRNA probes have a limit of detection of approximately 5 pg of purified viroid.

For a long time viroids have been detected by bioassay. Electrophoretic assay was introduced in 1976 for Potato spindle tuber. In the past months we developed this technique for Chrysanthemum stunt. Till now, the second Chrysanthemum viroid, Chrysanthemum chlorotic mottle was still detected by bioassay. Even under the best environmental conditions and especially high temperature and illumination the test is long (4-8 weeks) and not reliable. We demonstrate here that the electrophoretic assay is also very suitable for the detection of CChMV. When carried with slab gels stained with ethidium bromide it exhibits a high sensitivity and presents the same advantages, reliability and versatility, as observed with PSTV and CSV., Le Chrysanthème peut être infecté par deux viroïdes, le rabougrissement (CSV) et les marbrures chlorotiques (CChMV). L’indexage classique consiste en une transmission par greffe sur des variétés indicatrices sensibles : les variétés utilisées sont le plus généralement « Mistletoe » d’une part, « Deep Ridge » ou « Yellow Delaware » d’autre part, pour le rabougrissement et les marbrures chlorotiques respectivement. Dans les meilleures conditions d’environnement (28-30 °C, 15 000 lux et 85 p. 100 d’humidité), les symptômes caractéristiques n’apparaissent que plusieurs semaines après inoculation et le pourcentage des plantes inoculées développant des symptômes est faible notamment dans le cas du CChMV. Dans les dernières années, une technique d’électrophorèse a été développée d’abord pour l’indexage du viroïde des tubercules fusiformes de la Pomme de terre (PSTV), puis pour celui du rabougrissement du Chrysanthème. La technique utilisée pour le CSV a été appliquée au CChMV : elle est fondée sur l’extraction des acides nucléiques de la plante, l’élimination des gros RNA par LiC1 2M et la séparation des petits RNA par électrophorèse sur un gel de polyacrylamide à 5 p. 100 en plaque. La coloration des acides nucléiques par le bromure d’éthidium confère une grande sensibilité à cette technique qui peut détecter moins de 10 ng de RNA viroïdal. Ainsi le CChMV a pu être mis en évidence dans des échantillons végétaux très réduits : il suffit en effet d’un échantillon de 12 mg de jeunes feuilles ou de racines pour détecter le viroïde qui peut être également observé dans des échantillons de 100 mg de feuilles âgées ou de tiges. La technique électrophorétique d’indexage est également applicable à des jeunes plantes issues de cultures in vitro. Par ailleurs, l’indexage électrophorétique est plus faible que l’indexage biologique ; en effet, le viroïde est régulièrement mis en évidence dans des plantes inoculées par CChMV et ne présentant pas de symptômes. Les deux viroïdes, CSV et CChMV, qui ont en gel de polyacrylamide à 5 p. 100 la même mobilité, ne peuvent être différenciés par la technique électrophorétique qui, en contre-partie, présente l’avantage de pouvoir, dans les mêmes conditions, déceler dans un contrôle de routine aussi bien le CSV que le CChMV.

Recent progress in chrysanthemum viroid detection. Symposium on Chrysanthemum

Les effets du PSTV ont été étudiés sur plusieurs cultivars de pomme de terre infectés artificiellement et maintenus en serre isolée. Dans ces conditions de culture, l’infection a peu d’effet sur les organes aériens ; en revanche, on observe un effet très marqué sur la taille et les facultés germinatives des tubercules de certaines variétés, notamment « Belle de Fontenay ». L’évolution de l’infection dans la plante a été suivie tout au long du cycle végétatif, dans les feuilles, puis dans les tubercules. Le PSTV a été recherché par la technique d’hybridation moléculaire utilisant une sonde constituée d’un plasmide contenant une copie complète de PSTV et marquée au 3P2 par « nick translation ». La présence du PSTV peut être mise en évidence très précocement après l’inoculation et il existe une parfaite corrélation entre les résultats des tests effectués sur les parties aériennes et sur les tubercules. Cependant, une certaine hétérogénéité de la distribution du PSTV pose le problème de l’échantillonnage. L’intérêt et la sensibilité de la technique d’hybridation moléculaire (qui permet de détecter régulièrement 15 à 30 pg de viroïde) ont été confirmés. Cette méthode apparaît particulièrement adaptée à une détection précoce du viroïde aussi bien dans les organes aériens que dans les tubercules., Several potato cultivars were artificially inoculated. Under our culture conditions in an isolated greenhouse, PSTV appeared to have little effect on the above-ground part of the plant ; by contrast, it has marked effect on tuber production and quality : the size and germination of tubers of some cultivars were particularly affected (Belle de Fontenay). Spread of PSTV in the plant from the inoculation point was followed by sampling of leaves during the vegetation period, and subsequently of tubers. PSTV was detected by a molecular hybridization technique using a 32P-labelled probe consisting of a plasmid containing a full-length copy of PSTV. Presence of PSTV was detected as early as 9 days after inoculation and the results point to a good correlation between the results of the tests carried out on leaves and on tubers. Nevertheless uneven distribution of the viroid in leaves and tubers was observed, which poses the problem of sampling. The reliability and sensitivity of the molecular hybridization technique (which detects as low as 15-30 pg viroid) were confirmed : this technique appears to be perfectly adapted to early detection of PSTV in leaves and tubers of infected potato plants.

Summary A cloned 32 P-labelled cDNA probe to potato spindle tuber viroid (PSTV) was used to investigate the possibility of applying the spot hybridization technique to the identification of 2 virois PSTV and chrysanthemum stunt viroid (CSV). The best conditions for obtaining the highest sensitivity consisted of spotting heat-denatured samples onto the nitro-cellulose membrane, then hybridizing at 42 or 55°C in the presence of 50% formamide. The technique led to detection of 100–250 pg of purified viroid RNA and to identification of the viroid in a plant extract corresponding to 0.1 mg of infected leaves. These values demonstrated a clear advantage of the hybridization technique over the electrophoretic assay. A 3 H-labelled probe could be substituted for the 32 P probe, but it exhibited poor sensitivity due to the initially low specific radioactivity of the 3 H probe. Despite large sequence homologies, the PSTV 32 P-labelled probe hybridized only with CSV in purified solutions under non-stringent conditions, e. g. low temperature (42°C) and in the presence of relatively high amounts of CSV (50 mg). Therefore, spot hybridization appeared to be a more sensitive diagnostic technique than the electrophoretic assay, but with a high degree of specificity even with apparently closely related viroids.

We developed a dot-blot hydridization assay for the detection of Chrysanthemum stunt viroid (CSV) in Chrysanthemum plant samples. The probe, a recombinant plasmid containing a full-length monomeric cDNA copy of CSV, is labelled with (32P) by nick-translation. The influence of the hybridization conditions, of the sample denaturation technique and of the plant sap components on the final sensitivity has been studied. The optimized system, involving a formaldehyde denaturation step, allows the detection of as little as 5 pg of purified viroid. Under these conditions, 100 pg of pure viroid diluted in plant sap, or infected plant extract diluted 1:25 in healthy extract can be detected, showing the potential of this method for indexing of Chrysanthemum for CSV infection.

The two Chrysanthemum viroids, Chrysanthemum stunt (CSV) and Chrysanthemum chlorotic mottle (CChMV) are detected by polyacrylamide gel electrophoresis ; the electrophoretic assay is an adaptation to slab gels (PASGE) of the Morris and Smith's procedure previously applied to Potato spindle tuber viroid. Use of ethidium bromide staining, specific of double stranded nucleic acids, makes the method much more sensitive than after toluidine blue 0 staining or by U.V. scanning of cylindrical gels.Compared to the biological indexing the technique appears highly sensitive : this biochemical test allows CSV detection in samples as small as 10 mg plant material.By using PASGE the presence of viroids can be noticed earlier after infection than with the normal biological indexing : viroid RNA can be observed in plants which do not show any symptom.The importance of sampling is emphasized. Viroids are detected in any part of the infected Chrysanthemum plants. Nevertheless, viroids concentration is higher in the tips and the young leaves which are the best material for indexing.This technique has been applied to a large number of samples. It is simple, rapid, highly sensitive and reliable and consequently seems to be very appropriate to a large scale indexing.

Resume Les virus du groupe des Nepovirus possedent un genome bipartite. Dans le cas du sous-groupe du virus des anneaux noirs de la tomate (TBRV: tomato black ring virus), les RNA1 (2,5 × 10 6 d) et RNA2 (1,5 × 10 6 d) sont respectivement encapsides dans les particules B et M. Certaines souches de TBRV comme la souche S etudiee ici, qui se caracterise par un pouvoir pathogene eleve et une symptomatologie particuliere, possedent un troisieme RNA, le RNA3. L'existence de ce petit RNA est rattachee a la presence de deux nucleoproteines M′ et T′. Nous montrons ici que ce RNA est eliminable de la souche S sans que cette elimination entraine de modification de symptomes; il possede une incidence sur les proportions relatives des RNA genomiques. Sa replication necessite la synergie des deux acides nucleiques majeurs de la souche S, et ne peut etre assuree ni par des souches de TBRV dont le genome est different et ne contient pas naturellement de RNA satellite, ni par des souches pseudo-recombinantes contenant l'un des acides nucleiques de la souche S.

International audience

Ten years after the original reports demonstrating the feasibility of the detection of plant viruses and viroids by PCR-based techniques, a large body of literature describes the development of PCR primer pairs and of very sensitive assays for essentially all groups of viral pathogens. The tests developed show, in general, a sensitivity in great excess (up to several thousand-fold) of that reported for serological assays. Yet, the assays reported have so far and to a very large extent been confined to laboratory scale set-ups. Transition from this situation to the real-life, large scale application of these techniques will require further validation as well as development of automated or semi-automated pre- and post-PCR steps. Past and current achievements as well as future perspectives are discussed.

Candresse, T., Cambra, M., Dallot, S., Lanneau, M., Asensio, M., Gorris, M. T., Revers, F., Macquaire, G., Olmos, A., Boscia, D., Quiot, J. B., and Dunez, J. 1998. Comparison of monoclonal antibodies and polymerase chain reaction assays for the typing of isolates belonging to the D and M serotypes of plum pox potyvirus. Phytopathology 88:198-204. Plum pox potyvirus (PPV) isolates may be divided into four groups separated by serological, molecular, and epidemiological differences. Monoclonal antibodies specific for the two major groups of isolates, represented by the D and M serotypes of the virus, have been obtained. Polymerase chain reaction (PCR)-based assays allowing the direct detection and differentiation of PPV isolates have also been developed. We now report on a large-scale comparison of these two typing approaches. The results obtained show an overall excellent correlation between the results obtained in indirect double-antibody sandwich enzyme-linked immunosorbent assay using PPV-D‐ and PPV-M‐specific monoclonal antibodies and those derived from either specific PCR assays or restriction fragment length polymorphism analysis of PCR fragments. Without exception, all isolates reacting positively with the PPV-M‐specific m onoclonal antibody were found to belong to the M serotype using the PCRbased assays, while 51 out of 53 isolates recognized by the D-specific monoclonal antibodies belonged to the D serotype according to the PCR typing results. However, failure to react with a specific m onoclonal antibody did not prove as effective a predictor of the serotype of the isolate analyzed. In a few cases, the results obtained with the various techniques diverged, indicating low level variability of the epitopes rec ognized by the serotype-specific monoclonal antibodies. Isolates belonging to the two minor groups of PPV (El Amar and Cherry) also gave divergent results, indicating that the current typing assays are not suited for the analysis of such isolates.