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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.

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