The First Report of Tomato spotted wilt virus on Gerbera and Chrysanthemun in Venezuela.

Gerbera (Gerbera jamesonii) and Chrysanthemum (family Asteraceae) are two of the top 10 cut flowers of the world, with great commercial value. Since 1998, Venezuela began a floral industry to produce and export fresh cut gerbera and chrysanthemum, with 40% of nurseries concentrated in Altos Mirandinos (Miranda State, north central region of the country). For the past 2 years, greenhouse-grown gerbera and chrysanthemum have been observed displaying symptoms resembling those associated with tospoviruses. Symptomatic plants showed concentric rings, irregular chlorotic blotches, and deformation on leaves. Disease incidence was estimated at 30%. Mechanical inoculation with extracts of symptomatic leaves reproduced the typical concentric ring symptoms on indicator plants Arachis hypogaea L. cv. San Martín, Capsicum chinense, and G. jamesonii 6 to 15 days after inoculation. In initial tests, leaves from each 30 symptomatic gerbera and chrysanthemum species from several greenhouse facilities in Altos Mirandinos reacted positively when tested by DAS-ELISA with polyclonal antisera (ATCC, Rockville, MD) raised against Tomato spotted wilt virus (TSWV). Total RNA was extracted with the RNeasy Plant Mini kit (QIAGEN, Hilden, Germany) from two gerbera and two chrysanthemum ELISA-positive samples. The TSWV coat protein gene was amplified by conventional reverse transcription (RT)-PCR using primers CP1 TSWV (TTAACTTACAGCTGCTTT) and CP2 TSWV (CAAAGCATATAAGAACTT) (1). A single DNA product of ~823 bp was amplified from all samples. RT-PCR products were directly sequenced in both orientations and sequences were deposited in GenBank (Accession Nos. KF146700 and KF146701 derived from chrysanthemum, KF146702 and KF146703 derived from gerbera). The resulting sequences showed over 99% identity with each other. and were found to be closely related (over 99%) with TSWV isolates deposited in GenBank originating from different hosts from France (FR693058, FR693055), Montenegro (GU339506, GU339508, GU355940), Italy (HQ830187), New Zealand (KC494501), South Korea (KC261967), and the United States (AY744476). To our knowledge, this is the first confirmed report of TSWV infecting gerbera and chrysanthemum in Venezuela. The relatively widespread occurrence of TSWV in Miranda State underscores the need for systematic surveys to assess its incidence and impact on ornamental crops so that appropriate management tactics can be developed. Reference: (1) R. A. Mumford et al. J. Virol. Methods 57:109, 1996.

Genetic variation of papaya ringspot virus in Venezuela.

The genetic variation of papaya ringspot virus (PRSV) in Venezuela was estimated by single strand conformation and nucleotide sequence analyses of two genomic regions of twenty-six isolates. These analyses showed that mutation, virus movement, selection, mixed infections and recombination contributed to shape the genetic variation observed. Phylogenetic analysis indicated that Venezuelan isolates were within a clade composed of isolates from the Americas and Australia. The genetic diversity of these isolates was sufficiently large that it must be taken into account when designing control strategies such as transgenic resistance and cross-protection.

First Report of Tomato yellow leaf curl virus in Venezuela.

Tomato yellow leaf curl virus (TYLCV), a member of the family Geminiviridae, is a serious production constraint to tomato worldwide. In the new world, the virus had been identified as the causal agent of tomato yellow leaf curl disease in the Caribbean countries of the Dominican Republic, Cuba, and Guadeloupe and also in Florida and the Yucatan Peninsula (1). Molecular data from these TYLCV isolates identified the virus as the TYLCV prototype from Israel. During April 2004, tomato plants showing symptoms such as chlorotic leaf edges, upward leaf cupping, leaf mottling, and reduced leaf size indicative of TYLCV were observed in commercial fields in Zulia state, Venezuela. Whiteflies (Bemisia tabaci Gennadius) were present in the field and appeared to be associated with the disease. Leaf samples from nine symptomatic plants were collected and brought to the lab at Instituto Venezolano de Investigaciones Científicas (IVIC) for further analyses. Geminivirus infection of samples was confirmed by PCR amplification with the degenerate primer pair PAL1v1978 and PAR1c494 (2). TYLCV coat protein gene-specific primers KL04-06_TYLCV CP F and KL04-07_TYLCV CP R (3) were used to confirm the diagnosis. These primers amplified the expected 842-bp PCR product from the nine symptomatic samples. One of the resulting amplicons was cloned into the pCR-TOPO vector (Invitrogen, Carlsbad, CA) and sequenced (GenBank Accession No. DQ302033). Sequence comparison with those available in the NCBI database indicated that the sequenced portion of the genome shared 99% nucleotide identity with the TYLCV mild strain from Portugal (GenBank Accession No. AF105975) and 98% nucleotide identity with the TYLCV mild strain from Spain (GenBank Accession No. AF071228), TYLCV Israel isolate (GenBank Accession No. AM234066), and TYLCV Mexico isolate (GenBank Accession No. DQ631892). To our knowledge, this is the first report of TYLCV infecting tomato crops in South America. Further studies are needed to clarify how TYLCV has been introduced into Venezuela. References: (1) J. E. Polston and P. K. Anderson. Plant Dis. 81:1358, 1997. (2) M. R. Rojas et al. Plant Dis. 77:340, 1993. (3) K. S. Ling et al. Plant Dis. 90:379, 2006.

First Report of Celery mosaic virus Infecting Celery in Venezuela.

Celery mosaic virus (CeMV) is a significant pathogen of celery (Apium graveolens) worldwide (1). In 2005, in a produce market located in Los Salias, Miranda, celery plants with mottling and leaf malformation were noticed. Electron microscopic analysis of leaf-dip preparations from three symptomatic samples revealed flexuous viral particles that were 750 nm long. Infected cells contained pinwheel inclusions typical of those associated with potyvirus infection. Inoculation of healthy celery plants with leaf extracts from four symptomatic plants produced symptoms identical to those first observed. A survey of five produce markets in Miranda was conducted to determine the prevalence of virus infection in celery using serological and molecular analyses. Mottling and malformation of celery leaflets were observed in all the markets visited. Symptoms were noted in all five markets in each of three visits during a 3-month period. A total of 125 postharvested symptomatic plants were collected from five markets on March 29, 2005 and tested for CeMV using double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with antiserum provided by F. Rabenstein, BAX, Aschersleben, Germany. Of the 125 samples collected during the survey, 53% were ELISA positive. Twenty ELISA-positive samples were also tested using reverse transcription-polymerase chain reaction (RT-PCR) with general primers for the family Potyviridae (2). All 20 samples produced an amplicon of the expected size (1.7 kbp) after RT-PCR. Amplicons from three samples were cloned into the pCR-TOPO vector (Invitrogen, Carlsbad, CA). Sequence analysis of one clone revealed more than 98% nt to a CeMV isolate from Australia (GenBank Accession No AF203532). To our knowledge, this is the first report of CeMV in Venezuela. Our results suggest that the disease may be widely spread on celery crops growing in the areas surrounding produce markets in Miranda State. References: (1) A. Brunt et al. Viruses of Plants. CAB International, Wallingford, Oxon, UK. 1996. (2) J. Chen et al. Arch. Virol. 146:757, 2001.

Natural Infection of Canavalia ensiformis with Tobacco mosaic virus in Venezuela.

Jack bean (Canavalia ensiformis) is a valuable green manure and cover-crop species. In late summer of 2002, jack bean plants showing severe stunting, leaf mosaic, mottling, distortion, and general yellowing were observed in fields located in Maracay, Aragua State, Venezuela. Sap from symptomatic leaves was used to mechanically inoculate healthy jack bean, and field symptoms were successfully reproduced. Similar inoculations on Nicotiana tabacum var. Sansum resulted in mosaic symptoms and leaf distortion. Electron microscopic examination of leafdip preparations showed filamentous rods resembling those of a tobamovirus. Tobacco mosaic virus (TMV) was specifically identified with TMV-specific polyclonal antibody (PVAS-958, ATTC) in enzyme-linked immunosorbent assay. Sequence analysis of a coat protein gene (CP) fragment amplified using reverse transcription-polymerase chain reaction (RT-PCR) with primers TMV-CP-F and TMV-CP-R (1) from total RNA confirmed the diagnosis. Nucleotide and deduced amino acid sequences of a 450-bp region of the RT-PCR product were 96 to 99% and 98 to 100% identical, respectively, to the TMV CP gene in GenBank Accession Nos. J02415 and X68110. On the basis of foliar symptoms, incidence of TMV in jack bean was more than 50% in this experimental field. The source of infection is not known. Because TMV is reported to be seedborne in many other plant species, testing jack bean seed stocks for TMV infection could have important implications on the future control of the virus. To our knowledge, this is the first report of natural infection of jack bean by a tobamovirus. Reference: (1) N. J. Spence et al. Eur. J. Plant Pathol. 107:633, 2001.

First Record of Turnip mosaic virus Infecting Watercress in Venezuela.

During 2001, watercress (Rorippa nasturtium-aquaticum) plants displayed in vegetable markets located in the district capital, Caracas, showed severe leaf distortion, chlorosis, and mosaic. Viral etiology was suspected, and several plants were brought to the lab for further analyses. Electron microscopic studies of leaf-dip preparations from symptomatic samples revealed flexuous viral particles 750 nm long. Infected cells contained pinwheel inclusions and scrolls typical of those associated with infection by a potyvirus. The virus was sap-transmitted back to watercress, which developed symptoms identical to those first observed. Disease symptoms were also reproduced on Nicotiana benthamiana using mechanical inoculation with watercress-infected sap. All samples were tested by enzyme-linked immunosorbent assay, using polyclonal antibodies (Agdia, Elkhart, IN) to Turnip mosaic virus (TuMV), the only potyvirus that has been found infecting watercress plants (1). TuMV was detected in 88% of 100 samples taken from 18 markets on a single day. Symptoms were noted in every market when visited once a month during a 6-month period. The high percentage of TuMV-infected watercress in the 18 local markets (all 10 to 11 km from watercress fields) makes further epidemiological studies desirable. To our knowledge, this is the first report of TuMV infection of watercress in Venezuela. References: (1) A. Brunt et al. Viruses of Plants. CAB International, Wallingford, Oxon, UK. 1996.

Detection of Poinsettia mosaic virus Infecting Poinsettias (Euphorbia pulcherrima) in Venezuela.

Poinsettia mosaic virus (PnMV), a putative member of the tymoviruses, was detected in several cultivars of vegetatively propagated poinsettias grown in commercial nurseries in Estado Miranda, Venezuela. Symptoms associated with the affected plants consisted of severe mottling and distortion of leaves and bracteoles. The suspect virus was mechanically transmitted to Nicotiana benthamiana. Leaf extracts and thin sections of affected leaf tissue were analyzed by transmission electron microscopy. Spherical virus particles (30 nm diameter) were observed in samples from symptomatic poinsettia plants. Ultrastructural analyses of virus-infected cells revealed aggregates of virus particles in the cytoplasm and central vacuole. The virus was purified twice from infected N. benthamiana, resulting in yields as high as 12 mg/100 g. Dissociated coat protein contained a single 24-kDa protein species. The virus was not serologically related to Carnation mottle, Bean rugose mosaic, Cowpea mosaic, Cucumber mosaic, Pea enation mosaic, Prunus necrotic ringspot, Apple mosaic, Tobacco streak, Maize rayado fino, Tomato ringspot, Bean southern mosaic, Sowbane mosaic, Andean potato latent, Belladona mottle, Scrophularia or Turnip yellow mosaic viruses, but did react positively in enzyme-linked immunosorbent assay and western blot analysis with antiserum (ATCC PVAS-476) to PnMV. Based on these results, the virus is considered to be PnMV. To our knowledge, this is the first report of PnMV infecting poinsettias in Venezuela.

Properties of a previously undescribed supercoiled filamentous virus infecting papaya in Venezuela.

Supercoiled filamentous virus particles with lengths of 400 to 700 nm and 3 nm wide were isolated from leaves of Carica papaya L. plants showing a mild yellowing between the veins. The morphological properties of this virus resemble those of tenuiviruses. However, it was serologically unrelated to three of the five definitive members of this group of plant viruses and had biochemical features quite different from tenuiviruses. Therefore, the virus described here is possible an unreported new virus infecting papaya for which the name of papaya mild yellow leaf virus (PMYLV) is proposed. PMYLV was mechanically transmitted to papaya and to several Cucurbitaceae species. Virus particles sedimented as one component in sucrose density gradients, containing one molecule of ssRNA with an apparent size of 6400 nucleotides which constitutes 5% of the particle weight. The buoyant density of PMYLV was 1.26 g/cm3 in cesium chloride equilibrium gradients, and the virus coat protein consisted of a single polypeptide with mol. wt. of c. 39 kDa. Estimated virus yield in purified preparations was 2.6 g/kg leaf tissues. An antiserum was produced with a titer of 1:1500. Ultrastructural observations of PMYLV-infected leaf tissues showed crystalline aggregates of virus particles, closely associated with electron dense amorphous inclusion bodies only within xylem cells.