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 Cucumber mosaic virus and Turnip vein-clearing virus in Dichondra repens in France, Italy, and China.

During surveys of Dichondra repens (kidneyweed, family Convolvulaceae) turfs in public gardens of the Franco-Italian Riviera from 1993 to 2003, leaf mosaic and yellow ringspot symptoms have been observed in Antibes, Menton, Nice, and Vallauris (France) and San Remo and La Mortola (Italy). Isolates from these six locations and from two locations in China (Shanghai and Kunming) have revealed the presence of Cucumber mosaic virus (CMV) based on the behavior of a range of manually inoculated plants (1), the observation of 30 nm isometric particles in semipurified extracts of inoculated Nicotiana tabacum 'Xanthi' plants with the electron microscope, and positive reactions in double antibody sandwich (DAS)-ELISAs with specific polyclonal antibodies. All isolates were shown to belong to group II of CMV isolates (3) by double-immunodiffusion analysis. CMV was previously identified in D. repens in California in 1972 (4). Following isolation from local lesions on Vigna unguiculata and multiplication in 'Xanthi' tobacco plants, two of the isolates were used to inoculate seedlings of D. repens manually or by Aphis gossypii aphids. Two months later, all inoculated plants showed symptoms similar to those previously observed and were positive in DAS-ELISA. In 2000, a D. repens sample collected in Antibes showing similar symptoms as above, induced necrotic local lesions in inoculated 'Xanthi' plants in 48 h, followed by systemic mosaic symptoms typical of CMV, therefore revealing the presence of a second virus. That virus was separated from CMV in apical, noninoculated leaves of Chenopodium quinoa and then used to inoculate a range of test plants. It was infectious in most plants of the families Solanaceae (including Cyphomandra betacea) and Brassicaceae, together with in Chenopodium amaranticolor, C. quinoa, Claytonia perfoliata, Convolvulus spp. 'Belle de jour', Digitalis purpurea, Gomphrena globosa, Ocimum basilicum, Plantago lanceolata, and Valerianella olitoria. It induced asymptomatic systemic infections in D. repens. Numerous, rod-shaped, 300 nm long particles were observed in sap extracts of infected plants with the electron microscope, suggesting the presence of a tobamovirus. A set of primers polyvalent for tobamoviruses (2) allowed the amplification of a DNA product of approximately 800 bp through reverse transcription-PCR performed with total RNA extracts from inoculated 'Xanthi' plants. The DNA product was cloned and sequenced (GenBank Accession No. EU927306) revealing that the virus belonged to a tobamovirus lineage including Ribgrass mosaic virus and viruses infecting cruciferous plants (Turnip vein-clearing virus [TVCV] and Youcai mosaic virus) and was closest to TVCV (95% amino acid identity; GenBank Accession No. NC_001873). To our knowledge, this is the first report of TVCV in D. repens. References: (1) L. Cardin et al. Plant Dis. 87:200, 2003. (2) A. Gibbs et al. J. Virol. Methods 74:67, 1998. (3) M. J. Roossinck. J. Virol. 76:3382, 2002. (4) L. G. Weathers and D. J. Gumpf. Plant Dis. Rep. 56:27, 1972.

First Report of Passiflora chlorosis virus in Bituminaria bituminosa in Europe.

Bituminaria bituminosa (L.) Stirton (pitch trefoil) is a perennial legume endemic to the Mediterranean Basin used as forage in arid areas and for stabilization of degraded soils. Mosaic and chlorotic ringspot symptoms have been observed in leaves of B. bituminosa in the Provence-Alpes-Côte d'Azur and Rhône-Alpes regions (France), Liguria (Italy), and Spain since 1975. In crude leaf extracts from more than 50 samples of diverse geographical origins, flexuous particles 680 to 720 nm long and 12 nm wide and pinwheel-like inclusions have been observed with the electron microscope, suggesting infection with a member of the family Potyviridae. The presence of a virus was confirmed by the use of potyvirus-polyvalent ELISA reagents (Potyvirus group test; Agdia, Elkhart, IN) and by the amplification of a DNA fragment of the expected size (≈1,650 bp) with extracts of isolates from different locations using reverse transcription (RT)-PCR with primers specific to members of the Potyviridae (3) corresponding to the 3' end of the virus genome. The amplified fragment of an isolate from Coaraze (Alpes Maritimes Department, France) was cloned and two cDNA clones corresponding to this amplicon were sequenced (GenBank Accession Nos. EU334546 and EU334547). These two sequences facilitated development of new primers (5'-AAARGCRCCCTATATAGCAG-3' and 5'-TATAAAGGTAACGCTAGGTGG-3') to specifically amplify and sequence the coat protein (CP)-coding region of isolates of the virus from five additional French locations. The amino acid sequences of the CP amplicon were more than 96% identical among the French isolates. Comparison with other virus sequences with the BLASTn program revealed that these isolates belonged to the same species as the potyvirus Passiflora chlorosis virus (2), with 89 to 90% and 95 to 97% identity at the nucleotide and amino acid levels, respectively, for the CP-coding region (1). The host range of the virus was evaluated by manual inoculation with the Coaraze isolate and was found to be very narrow. No symptoms and no infections were obtained in Capsella bursa-pastoris, Capsicum annuum, Claytonia perfoliata, Cucumis melo, Cucumis sativus, Cucurbita pepo, Datura stramonium, Gomphrena globosa, Medicago sativa, Nicotiana benthamiana, N. glutinosa, N. tabacum, Ocimum basilicum, Petunia hybrida, Phaseolus mungo, Physalis peruviana, Pisum sativum, Psoralea glandulosa, Ranunculus sardous, Salvia splendens, Solanum lycopersicum, Trifolium repens, Vicia faba, Vigna unguiculata, or Zinnia elegans. Necrotic local lesions were observed in Chenopodium amaranticolor, C. quinoa, and in all eight cultivars of Phaseolus vulgaris tested. The virus was transmitted either manually or by the green peach aphid (Myzus persicae) to healthy B. bituminosa seedlings. Symptoms appeared in 10 to 15 weeks, and the virus was detected in the symptomatic plants by RT-PCR. To our knowledge, this is the first report of a virus infecting B. bituminosa. References: (1) M. J. Adams et al. Arch. Virol. 150:459, 2005. (2) C. A. Baker and L. Jones. Plant Dis. 91:227, 2007. (3) A. Gibbs and A. M. Mackenzie. J. Virol. Methods 63:9, 1997.

First Report of Pilidium concavum on Bergenia crassifolia in France.

Bergenia crassifolia (L.) Fritsch (elephant's ears or Siberian tea) (Saxifragaceae) is a perennial rhizomatous plant with pink flowers appearing at the end of winter. Since 1990, large, brown, and necrotic spots have been observed on numerous B. crassifolia plants at the University of Sciences in Nice, France. Spots appeared each year in the spring on newly emerged leaves and enlarged up to 1 to 3 cm in diameter during the summer, sometimes affecting more than half of the leaf surface. Leaves with spots were collected from May to November and placed in a humid atmosphere. Black, sessile, discoid conidiomata developed on the spots and exuded a pink, then brown, spore mass. When a mass was transferred onto a 1% malt agar medium, mycelium grew and then numerous, relatively spherical conidiomata (0.5 to 2.5 mm in diameter) developed and exuded a pink slimy mass, which contained many conidia. The mycelium grown at 24°C in the dark was scarce and pale, pink-beige. Under the light, the fungal culture was much darker with a fluffy mycelium and numerous conidiomata. The base of the conidiomata was dark; conidiophores were hyaline and showed little segmentation. Unicellular, cylindrical, fusiform conidia were hyaline, 5.4 to 8 µm long, and 1.4 to 1.9 µm wide. The morphology and size of conidia were comparable with previous descriptions of Pilidium concavum (Desm.) Höhn. (2,3). The ITS1-5.8S-ITS2 region of two isolates was amplified by PCR with primers PN3 and PN10 according to Mendes-Pereira et al. (1) and sequenced. The 421-nt sequence (GenBank Accession No. FM211810) was 100% identical to that of the P. concavum specimen voucher BPI 1107275 (GenBank Accession No. AY487094). P. concavum was reported to be on stored or rotting leaves or fruits of many dicotyledonous plants (2). To validate Koch's postulates, pieces of mycelium cultures with conidiomata (28 days old) were placed onto the upper surface of leaves of healthy B. crassifolia plants (10 to 12 pieces per plant). The leaf epidermis was previously wounded with a needle and a drop of melted paraffin was poured onto each piece of mycelium to prevent desiccation. Agar plugs without the fungus were placed similarly on wounded leaves of two control plants. Four inoculated and two control plants were incubated in growth chambers at either 24 or 18°C (16 h of light per day, 15,000 lx, 80% humidity). At 24°C, brown spots developed from 90% of the inoculation sites, whereas spots were observed for only 18% of the sites at 18°C. Such spots did not develop on control plants. After 2 months, healthy leaves as well as those with necrotic spots were put in humid chambers. Conidiomata formed after 4 weeks and exuded the same pink mass, which contained numerous conidia and from which the fungus was reisolated. Similar symptoms were also observed in several other locations in France and in botanical gardens in Akureyri (Iceland) and Métis (Canada), from which P. concavum was reisolated. To our knowledge, this is the first report of P. concavum on B. crassifolia. References: (1) E. Mendes-Pereira et al. Mycol. Res. 107:1287, 2003. (2) M. E. Palm. Mycologia 83:787, 1991. (3) A. Y. Rossman et al. Mycol. Prog. 3:275, 2004.

First Report of Potato virus Y in Nicotiana mutabilis in France.

Nicotiana mutabilis Stehmann & Semir is a recently described perennial plant species from southern Brazil that produces long floral stems with white to deep pink flowers and is used for its ornamental quality. In 2003, leaf mosaic symptoms were observed in all 30 N. mutabilis plants in a nursery in the south of France. Observation of crude sap preparations with the electron microscope revealed numerous flexuous particles, 700 to 730 nm long and approximately 11 nm wide, associated with "pinwheel"-like cytoplasmic inclusions, typical of the family Potyviridae. A range of plant species inoculated with extracts from five of the symptomatic plants showed reactions typical of Potato virus Y (PVY) (2), and the presence of the virus was confirmed by positive reactions in double-antibody sandwich (DAS)-ELISA with polyclonal antibodies raised against PVY. To test if PVY was responsible for the symptoms observed in N. mutabilis, an isolate was multiplied in N. tabacum cv. Xanthi plants after isolation from local lesions on Chenopodium amaranticolor and was then mechanically inoculated to 12 seedlings of N. mutabilis cv. Marshmallow. After 3 weeks, the 12 inoculated plants showed systemic vein clearing symptoms and PVY was detected by DAS-ELISA. Reverse transcription (RT)-PCR tests using PVY-polyvalent primers (5'-GATGGTTGCCTTGGATGATG and 5'-TAAAAGTAGTACAGGAAAAGCCA) covering the coat protein (CP) coding region amplified a single DNA fragment of the expected 900 bp from total RNA extracts from Xanthi plants inoculated with the five isolates. One of these DNA products was directly sequenced (GenBank Accession No. EU252529) and several accepted methods of phylogenetic analysis compared this sequence to 80 available PVY CP coding sequences and showed that the N. mutabilis PVY belonged to the C1 group (1). Similar to the other PVY strains in the C group, the N. mutabilis isolate was able to induce hypersensitive local lesions in leaves of potato genotypes carrying the Nc gene. However, contrary to the other characterized C1 isolates (1), it was unable to infect systemically cv. Yolo Wonder pepper plants. That peculiar behavior makes the N. mutabilis isolate a tool to identify the viral determinants controlling the host range of PVY. References: (1) B. Blanco-Urgoiti et al. J. Gen. Virol. 79:2037, 1998. (2) C. Kerlan. No. 414 in: Descriptions of Plant Viruses. CMI/AAB, Kew, Surrey, UK, 2006.

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.

First Report of Cucumber mosaic virus in Viola hederacea in France and Italy.

Viola hederacea Labill. (Australian violet or trailing violet), family Violaceae, is native to eastern Australia and used for its ornamental quality in humid conditions, especially in terrariums. Mosaic and chlorotic ringspots associated with a mild crinkling on leaves of V. hederacea were observed in gardens in southeast France and La Mortola, Italy in 2001 and 2003, respectively. These symptoms were different from the small chlorotic spots reported in the same species in Queensland, Australia, from which a rhabdovirus was isolated (1). In samples collected from both locations, presence of Cucumber mosaic virus (CMV) was suspected on the basis of the symptoms exhibited by a range of inoculated plants (2) and the observation of isometric particles of approximately 30 nm with the electron microscope in sap preparations. This was confirmed by positive reactions with CMV polyclonal antiserum (3) in double-antibody sandwich-ELISA. Because of the viscosity of the V. hederacea extracts, 0.01% (w/v) of pectolyase in phosphate buffer was used to grind the samples and the homogenate was kept at room temperature for 2 h. In double-immunodiffusion analysis, each isolate was shown to belong to the group II of CMV. Tomato strains D and To were used as positive controls for CMV groups I and II, respectively. The virus was transmitted by aphids (Myzus persicae) in a nonpersistent manner from infected Nicotiana tabacum cv. Xanthi-nc plants to healthy Xanthi-nc plants. To determine if CMV was responsible for the symptoms observed, the French and Italian isolates were multiplied in Xanthi-nc tobacco plants after isolation from local lesions on Vigna unguiculata and mechanically inoculated to 10 plants of V. hederacea. A systemic vein clearing developed on leaves 10 days after inoculation, followed by mosaic and crinkling. CMV was detected in each plant 3 weeks after inoculation. To our knowledge, this is the first report of CMV in V. hederacea in France and Italy. References: (1) D. H. Gowanlock and R. G. Dietzgen. Australas. Plant Pathol. 24:215, 1995. (2) L. Cardin et al. Plant Dis. 87:200, 2003. (3) J.-C. Devergne et al. Ann. Phytopathol. 10:233, 1978.

First Report of Cucumber mosaic virus in Echium candicans in France.

Echium candicans (Linn.) Herb. Banks (Pride of Madeira or Viper's Bugloss), family Boraginaceae, is a perennial shrub used in gardens for the ornamental quality of its deep blue inflorescences, especially in coastal areas near the Mediterranean Sea. Mosaic symptoms were observed in leaves of E. candicans in the Alpes Maritimes Department of southeastern France, St Jean Cap Ferrat in 1994, Menton in 2002, and Antibes in 2005. Symptoms exhibited in a range of inoculated plants including Nicotiana tabacum cvs. Xanthi and Samsun, Chenopodium quinoa, C. amaranticolor, Vigna unguiculata cv. Black, and Cucumis sativus cv. Poinsett were typical of Cucumber mosaic virus (CMV). Occurrence of CMV in one sample from each of the three localities was confirmed by the observation of isometric particles (approximately 30 nm) with the electron microscope in crude sap preparations from the infected plants, positive reactions in double-antibody sandwich (DAS)-ELISA to polyclonal antibodies raised against CMV (1), and the nonpersistent transmission of the virus from infected Xanthi to virus-free Xanthi plants by Myzus persicae. In double-immunodiffusion analysis, the three isolates were shown to belong to the CMV subgroup II (1,2). To determine if CMV was responsible for the symptoms observed, the isolate from Antibes was multiplied in Xanthi plants after isolation from local lesions on V. unguiculata and mechanically inoculated to 3-year-old plants of E. candicans tested to be free from CMV before the mechanical inoculation. One month after inoculation, mild mosaic symptoms were observed in young leaves and CMV was detected by DAS-ELISA in 10 of 10 inoculated plants. To our knowledge, this is the first report of CMV in E. candicans. References: (1) J.-C. Devergne and L. Cardin. Ann. Phytopathol. 7:225, 1975. (2) M. J. Roossinck. J. Virol. 76:3382, 2002.

First Report of Alfalfa mosaic virus on Rhamnus alaternus in France.

Rhamnus alaternus L. (evergreen buckthorn), family Rhamnaceae, is a small, hardy shrub from Mediterranean regions grown for its ornamental persistent green or variegated foliage. Chlorotic oak leaf or ringspot symptoms on R. alaternus leaves have been observed in southern France (Bellegarde, Gard department in 1998; Fanjaux, Aude department in 2000; and Saint-Jean-Cap-Ferrat, Alpes-Maritimes department in 2002). Samples from these three localities revealed the presence of Alfalfa mosaic virus (AMV) due to (i) the symptoms observed in an inoculated diagnostic host range previously described (1), (ii) observation of typical bullet-shaped virion particles of different sizes with transmission electron microscopy, (iii) nonpersistent transmission to Nicotiana tabacum cv. Xanthi-nc by Myzus persicae, and (iv) positive reaction in double-antibody sandwich-enzyme-linked immunosorbent assays (DAS-ELISA) to antibodies raised against AMV (gift of G. Marchoux). In addition, in Fanjaux, Viburnum tinus L. plants located close to the infected R. alaternus plants were also infected by AMV and exhibited typical intense calico mosaics (3). The close species, R. frangula L., was previously identified as a natural host for AMV in Italy (2). Following isolation from local lesions on Vigna unguiculata, the Fanjaux isolate was grown in cv. Xanthi-nc plants, where it induced a severe mosaic and stunting of the plants, and inoculated to 2-year-old virus-free R. alaternus plants either mechanically or with M. persicae (10 plants each). Plants were subsequently kept in an insect-proof greenhouse. At 8 and 12 months postinoculation, only one aphid-inoculated plant showed symptoms on young leaves and was AMV-positive in DAS-ELISA, while no mechanically infected plants were infected. This low infection level together with the rare observation of symptoms in natural conditions suggest that R. alaternus is not frequently infected by AMV. References: (1) L. Cardin and B. Moury. Plant Dis. 84:594, 2000. (2) F. Marani and L. Giunchedi. Acta Hortic. 59:97, 1976. (3) N. Plese and D. Milicic. Phytopathol. Z. 72:219, 1971.

Occurrence of Alternaria dichondrae, Cercospora sp., and Puccinia sp. on Dichondra repens in France and Italy.

Dichondra repens (kidneyweed or ponysfoot), family Convolvulaceae, is a perennial plant with persistent leaves and is grown alone or in association with turfgrass in subtropical and Mediterranean regions. Because of its prostrate growth habit, it does not need to be mowed. It is also used as a potted plant for house decoration. During surveys of lawns in public gardens of the Franco-Italian Riviera conducted from 1993 to 2003, we noticed 0.1- to 0.5-cm-diameter, brownish, necrotic spots on leaves of D. repens in Antibes, Cannes, Menton, Nice, and Vallauris (France) and in Arma di Taggia, Diano Marina, Imperia, La Mortola, Ospedaletti, San Remo, and Ventimiglia (Italy). Symptoms were more intense in the spring on young leaves but lesions remained all year on older leaves. Two species of fungal pathogens were frequently isolated from these spots. One fungus produced brown, erect conidiophores with brown, pear-shaped conidia and bifid, subhyaline beaks. Conidia formed singly, were composed of 8 to 10 cells with transverse and longitudinal crosswalls, and had one to four hyaline spurs frequently longer than the conidia. Conidia measured 90 to 260 × 16 to 29 µm. The pathogen, identified as Alternaria dichondrae (1), was previously characterized in Italy, New Zealand, and Argentina. The second fungus species produced clumps of erect, brown conidiophores with hyaline, filiform conidia composed of 10 to 20 cells. These conidia measured 90 to 310 × 3 to 3.5 µm. This fungus was identified as a Cercospora sp. (2), a genus not previously reported on D. repens. For both fungi, necrotic spots similar to those observed in natural infections were obtained after spraying a suspension of mycelium and conidia onto leaves of D. repens seedlings that had two to four expanded leaves that had been pricked with a pin. The plants were maintained under high humidity. Assays of mycelium growth on agar media containing various fungicides showed that 1 ppm of pyremethanil completely inhibited the growth of A. dichondrae, whereas a mixture of 10 ppm of diethofencarb and 10 ppm of carbendazine completely inhibited Cercospora sp. growth. Telia were also observed on the lower surface of D. repens leaves, sometimes in association with disease symptoms of A. dichondrae and Cercospora sp. Disease symptoms of the rust were yellowing and curling of the leaf surface with erect petiole, whereas healthy plants were prostrate with plane leaf surfaces. The two-celled teliospores had smooth cell walls, a single germinative pore per cell, and measured 32 to 34 × 12 to 13 µm with a thin unattached pedicel. This rust fungus was consequently classified in the genus Puccinia (2), also not previously reported as a pathogen of D. repens. It is possible that Poaceae plants such as Poa pratensis grown in association with D. repens were the inoculum source. Whereas A. dichondrae and Cercospora sp. do not induce severe diseases and are not widespread, the prevalence of Puccinia sp. tends to increase over time, requiring appropriate treatments to manage infected turf grasses. References: (1) P. Gambogi et al. Trans. Br. Mycol. Soc. 65:322, 1975. (2) G. Viennot-Bourgin. Les Champignons Parasites des Plantes Cultivées, Masson ed. Paris, 1949.

Occurrence of Mosaic Caused by Cucumber mosaic virus in Lobelia Hybrids in France and Italy.

In 2002, mosaic symptoms associated with yellowish ringspots were observed on leaves of a hybrid of lobelia (Lobelia spp.) grown in a public garden in Alsace (France). In 2003, similar symptoms were observed in Lobelia laxiflora in the Hanbury botanical garden (La Mortola, Italy) and the botanical garden of Nice (France). Cucumber mosaic virus (CMV) was identified in samples collected from the three locations on the basis of the following: symptoms exhibited by a host range of inoculated plants previously described (1); the observation of isometric particles (approximately 30 nm) with an electron microscope in crude sap preparations from inoculated plants and semipurified extracts of Claytonia perfoliata; and the positive reaction in double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA) to antibodies raised against CMV (2). In double-immunodiffusion analysis, each isolate was shown to belong to the group II strains of CMV (4). In these experiments, no differences were observed among the isolates collected. To test if CMV was responsible for the symptoms observed in Lobelia spp., an isolate from Alsace was grown in Xanthinc tobacco plants following isolation from local lesions on Vigna unguiculata and then mechanically inoculated to L. × speciosa cv. Compliment mix (10 plants), L. siphilitica (10 plants), L. inflata (Indian tobacco) (10 plants), L. erinus cvs. Crystal and Empereur Guillaume (5 plants), L. erinus pendula cvs. Saphyr and Cascade (5 plants), L. laxiflora (10 plants), and L. × gerardii cv. Vedrariensis (5 plants) and grown in a hydroponic system. Eight weeks postinoculation, all plants except L. laxiflora exhibited systemic mosaic and chlorotic ringspot symptoms on leaves and resulted in strong DAS-ELISA reactions for CMV, whereas mock-inoculated controls remained symptomless and virus free. Symptoms were particularly severe on L. siphilitica and L. × speciosa, but mild on L. inflata and L. × gerardii. Foliar mosaic symptoms appeared only 6 months postinoculation in 7 of 10 inoculated L. laxiflora plants. Only these plants were CMV positive using DAS-ELISA. No symptoms were observed in flowers of any plants infected with CMV. CMV has been previously reported in other species of the family Lobeliaceae including L. cardinalis, L. erinus, L. gracilis, and L. tenuior following natural or experimental infection (3) but Koch's postulates were not completed. This study validates that CMV is responsible for mosaic diseases in Lobelia spp., and shows that hybrids from L. cardinalis such as L. × speciosa and L. × gerardii also are susceptible to CMV. Mosaic symptoms in L. siphilitica and L. × speciosa are particularly damaging to their ornamental quality. Moreover, perennial plants such as L. laxiflora can be sources of CMV contamination by aphid transmission. References: (1) L. Cardin et al. Plant Dis. 87:1263, 2003. (2) J. C. Devergne and L. Cardin. Ann. Phytopathol. 7:225, 1975. (3) L. Douine et al. Ann. Phytopathol. 11:439, 1979. (4) M. J. Roossinck. J. Virol. 76:3382, 2002.

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.

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.

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.

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.

First Report of Alfalfa mosaic virus in Pachysandra terminalis in Europe.

Pachysandra terminalis (Buxaceae) was introduced from Japan to Europe in 1882. This ornamental plant is grown in northern Europe as a ground cover in shaded sites. Line patterns, more or less necrotic ringspots, and mosaic symptoms on leaves of pachysandra plants have been seen in public gardens in France (Strasbourg, Colmar, Mulhouse, and Nantes) and Germany (Freiburg im Breisgaü). Extracts of plant tissues obtained from these five sites were used for mechanical- and aphid-transmission experiments, enzyme-linked immunosorbent assays (ELISA) with antibodies directed toward a tomato strain of Alfalfa mosaic virus (AMV) (from G. Marchoux, INRA, France), and electron microscopy. All inoculations produced symptoms typical for AMV in Nicotiana tabacum cv. Xanthi-nc tobacco, Chenopodium amaranticolor, C. quinoa, Vigna unguiculata, Phaseolus vulgaris, Vicia faba, Pisum sativum, and Ocimum basilicum (2). Moreover, Medicago arborea, a new host for AMV, showed systemic mosaic on leaflets. On these and the original pachysandras, AMV was readily detected by ELISA. After isolation from three to four local lesions on Vigna unguiculata and further multiplication in tobacco, one isolate was purified. Bacilliform particles of three sizes, typical for AMV, were seen by electron microscopy. Transmissions of the strain to ELISA-negative pachysandras was achieved by mechanical inoculations (7 of 20 inoculated plants were ELISA positive) and by the aphid species Myzus persicae and Aphis craccivora (14 of 17 inoculated plants were infected). Symptoms were observed 2 months after inoculation; some of the plants remained symptomless but were AMV positive in ELISA. As early as 1970, similar symptoms were reported in pachysandra in New Jersey, and AMV was isolated from affected plants (1). However, inoculations of healthy pachysandra plants with AMV was not performed. Our results show the need for an AMV indexing protocol in the propagation of pachysandra to control its spread. References: (1) D. E. Hershman and E. H. Varney. Plant Dis. 66:1195, 1982. (2) E. M. J. Jaspars and L. Bos. 1980. Alfalfa mosaic virus. No. 229 in: Descriptions of Plant Viruses. Commonw. Mycol. Inst./Assoc. Appl. Biol., Kew, England.

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.

Serotype specificity of monoclonal antibodies to cucumber mosaic virus.

Ten monoclonal antibodies (McAbs) against cucumber mosaic virus (CMV) have been raised from fusion experiments performed after immunizing mice with different CMV antigens. Their reactivities with members of the three CMV serotypes, CMV-DTL, CMV-ToRS, and CMV-Co were tested in a double antibody sandwich format of enzyme immunosorbent assay (ELISA). Several of the McAbs were specific for different members of the CMV-DTL and CMV-ToRS groups while two allowed the detection of CMV-Co. By using a mixture of two McAbs, any member of the three major CMV serotypes could be detected in infected plant sap. One of the antibodies made it possible to discriminate between subunits and whole virions of CMV-D when it was used in ELISA simultaneously as coating antibody and as biotin-conjugate. McAbs were shown to be useful for quantifying the amount of CMV present in plant sap.

The effect of benzoic acid derivatives on Nicotiana tabacum growth in relation to PR-b1 production.

Using HPLC quantification, we have shown that benzoic acid derivatives stimulate PR-b1 protein synthesis in the leaf discs of Nicotiana tabacum Xanthi nc. The stimulation of PR-b protein synthesis during treatment with several benzoic acid derivatives is described for the first time in the root system of in vitro grown Nicotiana tabacum plantlets. In healthy in vitro grown plantlets the PR-b1 concentration is similar in roots and leaves (200 ng per gram of fresh material). During chemical treatment, however, the PR-b1 concentration increases to a lesser extent in roots than in leaves (10-fold higher in treated roots and 100-fold higher in treated leaves). Benzoic acid derivatives also have a detrimental effect on the growth of in vitro plantlets, which may be related to the accumulation of PR-b proteins.