First Report of Cucumber mosaic virus in Paeonia lactifera in France.

Chinese peony (Paeonia lactiflora Pall.), a hardy ornamental plant of the family Paeoniaceae cultivated in gardens and for cut flower production, is frequently infected by Tobacco rattle virus (TRV) in the field. The virus usually induces severe mosaic and chlorotic ringspot symptoms in the leaves, decreasing the commercial value of cut flowers. TRV is routinely detected by mechanical inoculation onto Nicotiana tabacum cv Xanthi, where it induces typical necrotic local ringspots in 3 to 7 days, followed by a reverse transcription (RT)-PCR test (2). In 2004, Xanthi test plants inoculated with sap extracts from 4 of 36 P. lactiflora cv. Odile plants grown in a field plot in the region of Hyères (southeast France) showed systemic mosaic symptoms in addition to the TRV-typical response. In each case, Cucumber mosaic virus (CMV) was detected by the reactions of a range of inoculated plants (1), the observation of 30 nm isometric particles in crude leaf extracts with the electron microscope, and by positive reactions in double antibody sandwich (DAS)-ELISAs with specific polyclonal antibodies. In double-immunodiffusion analysis, these isolates were shown to belong to the group II of CMV isolates (3). ELISA of the peony plants confirmed the presence of CMV and revealed two additional infected plants in the spring of 2006. Following isolation from local lesions on Vigna unguiculata and multiplication in Xanthi tobacco plants, one of the isolates was used to inoculate manually or with Myzus persicae aphids 10 CMV-free plants of P. lactiflora cv. Odile obtained from meristem culture. Three months postinoculation, only three of the aphid-inoculated plants were CMV positive by DAS-ELISA. No change was observed at 1 year postinoculation and no symptoms have been observed, even in CMV-infected plants. CMV appears to be latent in P. lactiflora, therefore detection of CMV before vegetative propagation of the plants is advised because of the risks of synergism for symptoms with other viruses such as TRV. To our knowledge this is the first report of CMV in peony. References: (1) L. Cardin et al. Plant Dis. 87:1263, 2003. (2) D. J. Robinson J. Virol. Methods 40:55, 1992. (3) M. J. Roossinck. J. Virol. 76:3382, 2002.

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. References: (1) J.-C. Devergne et al. Ann. Phytopathol. 10:233, 1978. (2) Y. Ignab and A. Putnaergle. Tr. Latv. S.-Kh. Akad. 118:27, 1977. (3) M. Navalinskiene and M. Samuitiene. Biologija 1:52, 1996. (4) D. J. Robinson. J. Virol. Methods 40:57, 1992.

Mosaic Symptoms Induced by Cucumber mosaic virus in Polygala myrtifolia in France and New Zealand.

Myrtle-leaf milkwort or sweet pea shrub (Polygala myrtifolia L.), family Polygalaceae, is a shrub from South Africa and is well adapted to Mediterranean-type conditions and used as an ornamental plant in gardens and pots or as cut flowers. During 2002 and 2003, mosaic symptoms and leaf distortion were observed in P. myrtifolia in Menton, Roquebrune-Cap Martin, Golfe Juan, and Antibes (Alpes Maritimes Department, France) in public gardens and potted plants. Occasionally, white streaks were observed in flowers. Cucumber mosaic virus (CMV) was identified in samples collected from the four locations on the basis of transmission to and symptoms exhibited by a range of diagnostic host plants (1), observation of isometric particles (≅30 nm) in crude sap preparations from the infected plants by electron microscopy, and positive reaction using double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA) with polyclonal antibodies raised against CMV (2). Each isolate was shown to be a group II CMV strain (3) using double-immunodiffusion analysis. During 2004, CMV was also detected using DAS-ELISA in P. myrtifolia samples collected in New Zealand (Christchurch, Akaroa, and Roturoa). To confirm that CMV was responsible for pathogenicity, the Menton isolate was isolated from local lesions on Vigna unguiculata, amplified in Nicotiana tabacum cv. Xanthi-nc, and then mechanically inoculated into 1-year-old P. myrtifolia, P. myrtifolia cv. Grandiflora, and P. myrtifolia cv. Compacta (synonymous to cv. Nana) plants. The D strain of CMV, a reference tomato strain from subgroup I (2), was used for comparison. All experimental plants were propagated from cuttings, grown hydroponically and all tested negative for CMV using DAS-ELISA prior to inoculation. At 12 weeks postinoculation, systemic symptoms were observed on leaves from all inoculated plants (10 plants per genotype for the Menton isolate and 5 plants per genotype for the D strain), except for two P. myrtifolia plants inoculated with the Menton isolate. CMV was detected in apical, noninoculated leaves using DAS-ELISA in all symptomatic plants. A total recovery from symptoms was observed in P. myrtifolia and P. myrtifolia cv. Grandiflora but not in P. myrtifolia cv. Compacta at 6 months postinoculation (mpi) in 7 of 15, 10 of 15, and 15 of 15 DAS-ELISA positive plants, respectively. At 7 mpi, the plants were pruned and planted in soil and at 8 mpi, CMV was detected using DAS-ELISA in most of the plants, and symptoms developed in a few stems of some of the plants. Tessitori et al. (4) described similar symptoms and have detected CMV in P. myrtifolia from Italy, but they did not reproduce the disease in healthy plants. Our results show that CMV is responsible for the symptoms observed and that both CMV subgroups are infectious in P. myrtifolia. Since P. myrtifolia is generally vegetatively propagated by cuttings, frequent CMV tests on the mother stock plants are recommended because of fluctuations in virus titer and symptom expression in some genotypes. To our knowledge, this is the first report of this CMV host in France and New Zealand. A voucher specimen will be deposited at the Station de Pathologie Végétale at INRA, Montfavet. References: (1) L. Cardin et al. Plant Dis. 87:1263, 2003. (2) J. C. Devergne and L. Cardin. Ann. Phytopathol. 7:225, 1975. (3) M. J. Roossinck. J. Virol. 76:3382, 2002. (4) M. Tessitori et al. Plant Dis. 86:1403, 2002.

Survey of Prunus necrotic ringspot virus in Rose and Its Variability in Rose and Prunus spp.

ABSTRACT A 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.

Enzyme-Linked Immunosorbent Assay Testing of Shoots Grown In Vitro and the Use of Immunocapture-Reverse Transcription-Polymerase Chain Reaction Improve the Detection of Prunus necrotic ringspot virus in Rose.

We developed and evaluated two different methods to improve the detection of the most prevalent virus of rose in Europe, Prunus necrotic ring-spot virus (PNRSV). Immunocapture-reverse transcription-polymerase chain reaction was estimated to be about 100 times more sensitive than double-antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) and showed an equivalent specificity. Based on the observation that PNRSV multiplies actively in young growing tissues (axillary shoots and cuttings), an in vitro culture method allowing rapid (about 15 days) and homogeneous development of dormant axillary buds with high virus titers was standardized. ELISA tests of these young shoots showed, in some cases, a 10(4) to 10(5) increase in sensitivity in comparison to adjacent leaf tissues from the rose mother plants. Between 21 and 98% (depending on the season) more samples were identified as positive by using ELISA on samples from shoot tips grown in vitro rather than on leaves collected directly from the PNRSV-infected mother plants. This simple method of growing shoot tips in vitro improved the confidence in the detection of PNRSV and eliminated problems in sampling appropriate tissues.

First Report of Alfalfa mosaic virus in Physostegia virginiana.

Physostegia 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. Reference: (1) L. Cardin and B. Moury. Plant Dis. 84:594, 2000.

First Report of Cucumber mosaic virus in Helleborus foetidus in France and Italy.

Helleborus 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. References: (1) J. C. Devergne and L. Cardin. Ann. Phytopathol. 7:225, 1975. (2) J. C. Devergne et al. Ann. Phytopathol. 10:233, 1978. (3) M. J. Roossinck. J. Virol. 76:3382, 2002.

First Report of Cucumber mosaic virus in Teucrium fruticans.

Teucrium fruticans (shrubby germander), family Lamiaceae, is a hardy shrub. Being drought tolerant, it is widespread in the Mediterranean area. Because it is readily propagated through cuttings, it is also planted in hedges. In 1997 and 2000, respectively, yellow chlorotic areas were observed on the foliage of T. fruticans in Saint Jean Cap Ferrat (France) and San Remo (Italy). These symptoms were distinct from those produced by a rust that frequently affects T. fruticans in these areas. Viruses from both locations were identified as Cucumber mosaic virus (CMV) based on the following: (i) symptoms after mechanical inoculation of Nicotiana tabacum cv. Xanthi nc, N. tabacum cv. Samsum, Chenopodium quinoa, C. amaranticolor, Vigna unguiculata cv. Black, and Cucumis sativus cv. Poinsett; (ii) the morphology of particles observed in electron microscopy of uranyl acetate stained leaf dips from tobacco; and (iii) positive result from leaves of diseased T. fruticans and mechanically inoculated host plants cited above based on enzyme-linked immunosorbent assay (ELISA) using CMV antisera. On tobacco cv. Xanthi nc, the French (F) and Italian (I) isolates first induced essentially necrotic rings on the inoculated leaves followed by the same systemic symptoms as described above. The two isolates were cloned from local lesions after two successive inoculations in V. unguiculata cv. Black, multiplied in tobacco, purified with the citrate-chloroform method, and stabilized with formaldehyde (1). The serotype determination was made by double immunodiffusion in agar gel with the CMV-D and CMV-To strains and homologous antisera (1,2). The formation of spurs and antigen-antibody lines indicated that both isolates belonged to the ToRS serotype (1). Thirty plants of T. fruticans cv. Azureum, first tested negative for CMV using ELISA, were mechanically inoculated with the F isolate (25 plants) and the CMV-D strain (five plants) and cultivated in a hydroponic system. Three months later, plants inoculated with the F isolate were positive for CMV using ELISA and displayed clear symptoms with chlorotic spots, which were sometimes ring-shaped. As plants mature, symptoms tend to disappear on young shoots. For the CMV-D strain, three plants of five were ELISA positive, but did not show any typical symptoms. This report demonstrates the infection of T. fruticans by CMV and the symptom induction by some CMV isolates. In September 2002, two CMV isolates were collected from T. fruticans in public gardens in Menton (France) and Genoa (Italy). These new isolates have the same characteristics as those described in this report. References: (1) J. C. Devergne and L. Cardin. Ann. Phytopathol. 7:225, 1975. (2) M. H. V. van Regenmortel. Adv. Virus Res. 12:207, 1966.