First report of cucumber mosaic virus in <em>Helleborus foetidus</em> in France and Italy

International audienceHelleborus foetidus L. (bear's foot) is a perennial plant from the family Ranunculaceae that is common in chalky soils of southern and western Europe. It is grown in gardens for its palm-shaped leaves and early flowers. In 1995, yellow-to-white oak leaf and line patterns in leaves of H. foetidus plants were observed in Hunawihr (Alsace, France). The same symptoms were observed in plants in Entrevaux, Biot, and Gourdon (Provence-Alpes-Côte d'Azur, France) in 2000 and 2001, in Triora (Liguria, Italy) in 2002, and on cv. Western Flisk in a nursery in Nice (Provence-Alpes-Côte d'Azur, France) in 2002. Samples collected from these six locations contained six isolates that were further characterized. Sap extracted from symptomatic plants was mechanically inoculated onto Nicotiana tabacum cvs. Xanthi-nc and Samsun, Chenopodium quinoa, C. amaranticolor, Vigna unguiculata cv. Black, and Cucumis sativus cv. Poinsett. Symptoms exhibited by the inoculated plants indicated infection by Cucumber mosaic virus (CMV). Sap extracted from symptomatic plants reacted positively in double-antibody sandwich-enzyme-linked immunosorbent assays (DAS-ELISA) to antibodies raised against CMV (2). Isometric particles (approximately 30 nm) were observed with an electron microscope in crude sap preparations from infected plants. Following purification of the suspect virus from infected N. tabacum (2) and treatment with formaldehyde (1), each isolate was shown to belong to group II of CMV strains (1,3) by double-immunodiffusion analysis. Following isolation from local lesions on V. unguiculata, the Hunawihr isolate was grown in cv. Xanthi-nc plants and back-inoculated to 2-year-old uninfected seedlings of H. foetidus by aphids (Myzus persicae) or mechanical transmission. Mechanical transmissions were also performed with sap extracted from cv. Xanthi-nc plants infected with the D strain, which belongs to group I of CMV strains (3). Three months postinoculation, symptoms previously described in the original plants were observed in 3 of 10 mechanically inoculated plants and in 2 of 14 aphid-inoculated plants (Hunawihr isolate), whereas no symptoms could be seen in any of the six plants inoculated with the D strain. On the basis of DAS-ELISA, 7 of 10 plants mechanically inoculated and 7 of 14 plants aphid inoculated with the Hunawihr isolate were infected with CMV, whereas 3 of the 6 plants inoculated with the D strain were infected with CMV. To our knowledge, this is the first report that H. foetidus is a natural host for CMV. Beyond the direct impact of the disease induced by CMV on H. foetidus, this perennial and widespread plant species can be an important reservoir of CMV

First report of Alfalfa mosaic virus in <em>Physostegia virginiana</em>

International audiencePhysostegia virginiana Benth. (false dragon head) is a perennial plant from the family Lamiaceae cultivated as an ornamental in gardens and for cut-flower production. In 2000, stunting of plants and yellow-to-brown ringspots on leaves were observed in cut-flower production in the Alpes Maritimes Department (southeast France). These symptoms greatly decreased the commercial value of the stems. The disease was attributed to Alfalfa mosaic virus (AMV) because extracts of infected plant tissues revealed typical bacilliform particles by electron microscopy, produced symptoms typical of AMV after inoculation of a range of previously described test plants (1), and reacted positively in enzyme-linked immunosorbent assays (ELISA) with antibodies raised to a tomato strain of AMV (from G. Marchoux, INRA, France). After isolation from single local lesions on Vigna unguiculata, the AMV isolate was multiplied in cv. Xanthinc tobacco, where it induced local and systemic ringspot symptoms. Infected Xanthinc plants served as sources of inocula for subsequent mechanical- and aphid (Myzus persicae)-transmission tests to healthy seedlings of P. virginiana (seeds from the botanic garden of Nancy, France; 36 plants for each inoculation procedure). Chlorotic and necrotic local lesions were observed in 25% of mechanically inoculated plants. Three months after inoculation, uninoculated leaves of all mechanically inoculated plants and 30.5% of aphid-inoculated plants tested positive for AMV based on ELISA. During the first year after inoculation, less than 10% of infected plants showed typical systemic symptoms. This proportion reached 40% during the second year. Recently, we observed similar symptoms in P. virginiana plants cultivated in public gardens in Intercourse (Pennsylvania), Toronto (Ontario, Canada) and Montreal (Quebec, Canada). Using ELISA, AMV was detected in symptomatic plants from these three additional locations

First report of tobacco rattle virus and cucumber mosaic virus in Phlox paniculata in France

Phlox paniculata L., a perennial plant from the family Polemoniaceae, is cultivated as an ornamental in gardens and for cut-flower production. In spring 2003, two types of symptoms were observed in P. paniculata plants grown for cut flowers on a farm in the Var department, France. Some plants showed a mild leaf mosaic while others showed leaf browning and delayed growth. In plants showing mild mosaic, Cucumber mosaic virus (CMV) was detected on the basis of the symptoms exhibited by a range of inoculated plants, the observation of isometric particles (approximately 30 nm) with the electron microscope in crude sap preparations from the infected plants, and the positive reaction in double-antibody sandwich (DAS)-ELISA to polyclonal antibodies raised against CMV (1). In double-immunodiffusion analysis, the five tested isolates were shown to belong to group II of CMV strains. To determine if CMV was responsible for the symptoms observed, one isolate was multiplied in Nicotiana tabacum cv. Xanthi-nc plants after isolation from local lesions on Vigna unguiculata and mechanically inoculated to 12 1-year-old P. paniculata plants. At 3 months post inoculation (mpi), all plants showed mild mosaic and CMV was detected by DAS-ELISA. In sap preparations from P. paniculata plants showing leaf browning symptoms, rod-shaped particles with two distinct sizes of 190 to 210 and 70 to 90 nm long, typical of those associated with tobraviruses, were revealed using electron microscopy. Local lesions typical of Tobacco rattle virus (TRV) were observed after inoculation of N. tabacum cv. Xanthi-nc, Chenopodium amaranticolor, and C. quinoa. Total nucleic acid preparations were prepared from symptomatic plants, and amplicons of the expected size (463 bp) were generated by reverse-transcription (RT)-PCR using primers specific to TRV RNA 1 (4). The nucleotide sequence of one amplicon was 93.6% identical to the sequence of a reference TRV isolate (GenBank Accession No. AJ586803). Twelve 1-year-old P. paniculata plants were mechanically inoculated with an extract of infected tissues from one symptomatic P. paniculata plant. TRV was detected 2 to 6 mpi in apical leaves of all inoculated plants by RT-PCR, although the plants did not express symptoms. Since no other pathogens were detected in the source plants, it is plausible that the lack of symptoms in back-inoculated plants is either due to a long incubation period or an interaction with particular environmental factors such as cold conditions. The survey of approximately 200 plants revealed that approximately 7, 10, and 1% were infected by TRV, CMV, or by both viruses, respectively. CMV and TRV were previously detected in P. paniculata in Latvian SSR and in Lithuania (2,3). These results show that sanitary selection of P. paniculata prior to vegetative propagation should include a screening for TRV and CMV infections

Survey of Prunus necrotic ringspot virus in rose and its variability in rose and Prunus spp

International audienceA survey for viruses in rose propagated in Europe resulted in detection of only Prunus necrotic ringspot virus (PNRSV) among seven viruses screened. Four percent of cut-flower roses from different sources were infected with PNRSV. Progression of the disease under greenhouse conditions was very slow, which should make this virus easy to eradicate through sanitary selection. Comparison of the partial coat protein gene sequences for three representative rose isolates indicated that they do not form a distinct phylogenetic group and show close relations to Prunus spp. isolates. However, a comparison of the reactivity of monoclonal antibodies raised against these isolates showed that the most prevalent PNRSV serotype in rose was different from the most prevalent serotype in Prunus spp. All of the 27 rose isolates tested infected P. persica seedlings, whereas three of the four PNRSV isolates tested from Prunus spp. were poorly infectious in Rosa indica plants. These data suggest adaptation of PNRSV isolates from Prunus spp., but not from rose, to their host plants. The test methodologies developed here to evaluate PNRSV pathogenicity in Prunus spp, and rose could also help to screen for resistant genotypes

Agrobacterium rhizogenes-mediated transformation of Prunus as an alternative for gene functional analysis in hairy-roots and composite plants

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699International audienceResistant rootstocks offer an alternative to pesticides for the control of soil pests. In Prunus spp., resistance loci to root-knot nematodes (RKN) have been mapped and a transformation method is needed to validate candidate genes. Our efforts have focused on the generation of transformed hairy-roots and composite plants appropriate for nematode infection assays. An efficient and reliable method using the A4R strain of Agrobacterium rhizogenes for the transformation of Prunus roots with an Egfp reporter gene is given. The rooting efficiency, depending on the genotypes, was maximal for the interspecific hybrid 253 (Myrobalan plum x almond-peach), susceptible to RKN, that was retained for subsequent studies. From the agro-inoculated cuttings, 72% produced roots, mainly at the basal section of the stem. Transformed roots were screened by microscope detection of Egfp fluorescence and molecular analyses of the integration of the transgene. The absence of residual agrobacteria in the plants was checked by the non-amplification of the chromosomal gene chvH. Egfp was expressed visually in 76% of the rooted plants. Isolated hairy roots in Petri dishes and composite plants (transformed roots and non-transformed aerial part) in soil containers were inoculated with the RKN Meloidogyne incognita. In both cases, root transformation did not affect the ability of the nematodes to develop in the root tissues. Our results showed that isolated hairy-roots can be used to validate candidate genes and the conditions in which composite plants offer a complementary system for studying the function of root genes in physiological conditions of whole plants are discussed

Transcriptome database resource and gene expression atlas for the rose

International audienceBackground: For centuries roses have been selected based on a number of traits. Little information exists on the genetic and molecular basis that contributes to these traits, mainly because information on expressed genes for this economically important ornamental plant is scarce. Results: Here, we used a combination of Illumina and 454 sequencing technologies to generate information on Rosa sp. transcripts using RNA from various tissues and in response to biotic and abiotic stresses. A total of 80714 transcript clusters were identified and 76611 peptides have been predicted among which 20997 have been clustered into 13900 protein families. BLASTp hits in closely related Rosaceae species revealed that about half of the predicted peptides in the strawberry and peach genomes have orthologs in Rosa dataset. Digital expression was obtained using RNA samples from organs at different development stages and under different stress conditions. qPCR validated the digital expression data for a selection of 23 genes with high or low expression levels. Comparative gene expression analyses between the different tissues and organs allowed the identification of clusters that are highly enriched in given tissues or under particular conditions, demonstrating the usefulness of the digital gene expression analysis. A web interface ROSAseq was created that allows data interrogation by BLAST, subsequent analysis of DNA clusters and access to thorough transcript annotation including best BLAST matches on Fragaria vesca, Prunus persica and Arabidopsis. The rose peptides dataset was used to create the ROSAcyc resource pathway database that allows access to the putative genes and enzymatic pathways. Conclusions: The study provides useful information on Rosa expressed genes, with thorough annotation and an overview of expression patterns for transcripts with good accuracy