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.

Complete nucleotide sequence of a putative new cytorhabdovirus infecting lettuce.

The full-length nucleotide sequence of the genomic RNA of a new cytorhabdovirus infecting lettuce was determined. Six open reading frames were found in the antigenomic sequence of the 12,926-nt negative-sense viral RNA genome. The genomic organisation was similar to that of lettuce necrotic yellows virus (LNYV), the type member of the genus Cytorhabdovirus: 3'-N-P-3-M-G-L-5', where N is the capsid protein gene, P the putative phosphoprotein gene, 3 a gene coding for a putative protein of unknown function, M the putative matrix protein gene, G the glycoprotein gene, and L the putative polymerase gene. Amino acid sequence comparison with the corresponding sequences of other rhabdoviruses revealed the closest relationship to LNYV, with identities ranging from 41% for the matrix proteins and 65% for the L polymerase proteins. These results indicate that this virus may be a member of a new cytorhabdovirus species, for which the name Lettuce yellow mottle virus (LYMoV) is proposed.

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 an Isolate of Pelargonium zonate spot virus in Commercial Glasshouse Tomato Crops in Southeastern France.

In summer 2000, symptoms similar to Pelargonium zonate spot virus (PZSV) were observed for the first time on tomato plants in southeastern France. The plants were from commercial glasshouse fresh-market crops. Symptoms observed were chlorotic mottling with bright yellow distinct rings on leaves and curved line patterns on stems. Fruit symptoms included chlorotic and necrotic spotting, marked concentric ring patterns, and distortions. Diagnosis was made from symptomatic leaves and fruits by mechanical inoculation on a set of host plants. Local chlorotic and necrotic lesions were observed on Chenopodium amaranticolor, C. quinoa, Cucumis sativus cv. Marketer, Cucumis melo cv. Vedrantais, Phaseolus vulgaris cv. Pinto, Vicia faba cv. D'Aguadulce, Vigna unguiculata cv. Black Eye, and systemic symptoms were observed on Capsicum annuum cvs. Yolo Wonder, Yolo Y, Florida VR2, and Criollo de Morelos 334, Datura stramonium, Lycopersicon esculentum cvs. Momor and Stevens, L. hirsutum (PI 134417 and PI 247087), Nicotiana benthamiana, N. clevelandii, N. tabacum cv. Xanthi nc, Ocimum basilicum cv. Latino, Petunia hybrida cv. Rose du ciel, and Physalis floridana. No reaction was observed on Pisum sativum cv. Douce Provence, Salvia splendens cv. Etna, or Zinnia elegans cv. Liliput. Symptoms on tomato of PZSV, Parietaria mottle virus (PMoV), and Tomato spotted wilt virus (TSWV) are similar, particularly those elicited in fruits. Therefore, the field samples were checked using double-antibody sandwich enzyme-linked immunosorbent assay against antisera of the type-strain of PZSV and tomato strain of PMoV and their homologous antigenes, which were supplied by D. Gallitelli and P. Roggero respectively, and our antiserum of TSWV. Electron microscopy of negatively stained preparations from leaves of tomato and D. stramonium showed that the sap contained very few paraspheric shaped particles, 26 to 29 nm in diameter. Three isolates collected from two different regions (Vaucluse and Bouches du Rhône) showed a very close serological relationship with the Italian type-strain of PZSV and tested negative against antisera of PMoV and TSWV. The French isolates were biologically different from the type-strain, but were similar to the Spanish strain of PZSV because they infected D. stramonium, N. benthamiana, O. basilicum, and V. unguiculata (2). Moreover, in transverse tissue sections, virions were not observed in the nucleus and tubular structures, unlike the Italian isolates, (1) but were present in the cytoplasm and particularly in the mesophyll cells. There are only a few records of the occurrence and distribution of PZSV in Mediterranean countries. References: (1) M. A Castellano and G. P Martelli. Phytopathol. Mediterr. 20:64, 1981. (2) M. Luis-Arteaga. Plant Dis. 84:807, 2000.

Cytological and molecular evidence that the whitefly-transmitted cucumber vein yellowing virus is a tentative member of the family Potyviridae.

Cucumber vein yellowing virus (CVYV) is widespread in cucurbits in the Middle East. CVYV has filamentous particles and is transmitted by Bemisia tabaci by the semi-persistent mode. It has not yet been assigned to a specific genus or family. Ultramicroscopic observations revealed numerous cylindrical cytoplasmic inclusions in melon and cucumber cells infected by CVYV isolates from Israel and Jordan. Depending on the section orientation, the inclusions appeared as pinwheels or as bundles. In addition, a 1.9 kb DNA fragment was amplified by RT-PCR from CVYV-infected plant extracts using primers designed to detect all potyvirids. Sequence comparisons with the amplified fragment indicated that CVYV is more closely related to Sweet potato mild mottle virus than to any other virus in the family Potyviridae: These results suggest that CVYV can be considered as a tentative new member of the genus Ipomovirus:, family Potyviridae:

Use of modified plum pox virus coat protein genes developed to limit heteroencapsidation-associated risks in transgenic plants.

Aphid transmission of a non-aphid-transmissible strain of zucchini yellow mosaic virus (ZYMV-NAT) occurs in transgenic plants expressing the plum pox potyvirus (PPV) coat protein (CP) gene. Heteroencapsidation has been shown to be responsible for this modification in the epidemiological characteristics of the infecting virus. In order to prevent this biological risk, several modified PPV CP constructs were produced that were designed to interfere with heteroencapsidation itself or to block aphid transmission of heteroencapsidated virions. These constructs were first expressed in Escherichia coli in order to check for the accumulation of pseudo-particles by electron microscopy. Virus-like particles (VLPs) were found with the full-length CP and with a PPV CP lacking the DAG amino acid triplet involved in aphid transmission. However, no VLPs were observed with CP lacking R220, Q221 or D264, amino acids known to be essential for the assembly of other potyvirus CPs. Transgenic Nicotiana benthamiana lines expressing the different PPV CP constructs were infected with ZYMV-NAT. Aphid transmission assays performed with these plants demonstrated that the strategies developed here provide an effective means of minimizing the biological risks associated with heteroencapsidation.

Characterization, nucleotide sequence and genome organization of leek white stripe virus, a putative new species of the genus Necrovirus.

White stripe is a disease affecting leek in France with which an isometric virus c. 30 nm in diameter is associated. The most evident symptom is the presence of white stripes on the leaves extending to the stem. Attempts to demonstrate transmission through the soil by sowing or transplanting leek in contaminated soil were unsuccessful. The virus was transmitted by sap inoculation to a narrow range of herbaceous hosts, all of which were infected only locally. Virus purification was from infected leek tissues, where it accumulated in large amounts, as demonstrated by ultrastructural observations. RNA was extracted from purified virus preparations and cDNA clones were prepared. The complete nucleotide sequence of the viral RNA was determined: The genome is 3,662 nucleotides long and contains five open reading frames (ORFs). The first (ORF 1) encodes a putative translation product of M(r) 23,803 (p24) and read through of its amber stop codon results in a protein of M(r) 82,625 (p83) (ORF 2). ORF 3 and ORF 4 encode two small polypeptides of M(r) 11,280 (p11) and M(r) 6,261 (p6), respectively. ORF 5 encodes the capsid protein of M(r) 27,460 (p27). The genome organization and sequence alignments with the corresponding products of necroviruses suggest that the virus isolated from leek is a new species in the genus Necrovirus, for which the name of leek white stripe virus (LWSV) is proposed.

[Optimization of gold immunolabeling techniques on ultrathin slides obtained from samples fixed with glutaraldehyde and osmium tetroxide and enclosed in epoxy resin]. / Optimisation de la technique de l'immunomarquage à l'or sur sections ultrafines réalisées à partir d'échantillons fixés à la glutaraldéhyde et au tétroxyde d'osmium et inclus dans une époxy-résine.

An immunogold labeling technique was carried out on plants infected with CMV, fixed with glutaraldehyde and osmium tetroxide and embedded in araldite CY 212. The effect of the type of support-film used, the resin and manipulations of the grids during immunogold steps, were studied and are discussed. The antigenic activity of virus was restored by treating the sections with sodium metaperiodate. The very high non-specific reactions observed with the support-film or with the resin were eliminated by adding powdered skimmed milk or non-purified albumin into the buffers. Purified bovine serum albumin (grade V) or chicken albumin (grade III to V) were inefficient in reducing this non-specific background.