R.A.A. van der Vlugt

First report of pepino mosaic virus on tomato.

Early in 1999 a new viral disease occurred in protected tomato (Lycopersicon esculentum) crops in the Netherlands. Infected plants showed yellow leaf spots and mosaic. Transmission electron microscopic analysis revealed particles typical of potexviruses. Only three potexviruses have been reported to infect solanaceous crops: Pepino mosaic virus (PepMV), Potato aucuba mosaic virus (PAMV), and Potato virus X (PVX). Inoculation of test plants and serological tests showed that the new virus clearly differed from PAMV and PVX. Immuno-electron microscopy with antiserum to PepMV (1), the original PepMV isolate, and the virus from tomato showed decoration titers of 1:800 (homologous) and 1:400, respectively. Neither virus reacted with antiserum to PVX, nor did PVX react with antiserum to PepMV. Results of host plant analysis with 17 plant species mostly resembled those expected for PepMV. Nucleotide sequence alignment of DNA fragments obtained by reverse-transcriptase polymerase chain reaction with a specific primer set for potexviruses, directed against the RNA polymerase region, showed 93% identity between PepMV and the virus from tomato, while homologies with PVX, PAMV, and other potexviruses were <60%. Results indicate that the potexvirus in tomato is PepMV. PepMV was first found in pepino (Solanum muricatum) in Peru in 1974 and described by Jones et al. in 1980 (1). This is the first report of a natural infection of tomato by PepMV. Reference: (1) R. Jones et al. Ann. Appl. Biol. 94:61, 1980.

Taxonomic relationships between distinct potato virus Y isolates based on detailed comparisons of the viral coat proteins and 3'-nontranslated regions

SummaryDetailed comparisons were made of the sequences of the coat protein (CP) cistrons and 3′-nontranslated regions (3′-NTR) of 21 (geographically) distinct isolates of potato virus Y (PVY) and a virus isolate initially described as pepper mottle virus (PepMoV). Multiple sequence alignments and phylogenetic relationships based on these alignments resulted into a subgrouping of virus isolates which largely corresponded with the historical strain differentiation based on biological criteria as host range, symptomatology and serology. Virus isolates belonging to the same subgroup shared a number of characteristic CP amino acid and 3′-NTR nucleotide residues indicating that, by using sequences from the 3′-terminal region of the potyvirus genome, a distinction could be made between different isolates of one virus species as well as between different virus species. RNA secondary structure analysis of the 3′-NTR of twelve PVY isolates revealed four major stem-loop structures of which, surprisingly, the loop sequences gave a similar clustering of isolates as resulting from the overall comparisons of CP and 3′-NTR sequences. This implies a biological significance of these structural elements.

Pepino mosaic virus.

Pepino mosaic virus (PepMV) is a relatively new plant virus that has become a signifi cant agronomical problem in a relatively short period of time. It is a member of the genus Potexvirus within the family Flexiviridae and is readily mechanically transmissible. It is capable of infecting tomato (Solanum lycopersicum) and other Solaneceous host plants. Since its description in 1980 from pepino plants (Solanum muricatum) collected in 1974 in Peru, the virus remained unknown for a long time until it manifested itself in commercial tomato crops in Europe in 1999. Since then the virus has been reported worldwide and the disease it causes has become important in commercial tomato production. Since 1999, new strains of the virus have been described which diff er from the original pepino isolate. The fast spread of the virus and the appearance of mixed infections with the new strains may play an important role in the increase of the agricultural importance of this viral disease

Nucleotide sequence of the 3' terminal region of the genome of four Lettuce mosaic virus isolates from Greece and Yemen

SummaryLettuce mosaic virus (LMV) is an economically important Potyvirus causing a severe disease of commercial lettuce crops. Based on molecular data, three phylogenetic groups of isolates have previously been discriminated, reflecting their geographical origin (Western Europe-California, Greece, or Yemen). Sequence information for the entire coat protein domain was only available for one of the Western Europe-California phylogenetic group. We have now sequenced the 3′ terminal region of the genome LMV-Gr4, -Gr5 and -GrB, isolates which belong to the Greek phylogenetic group and of LMV-Yar, the sole known representative of the third LMV phylogenetic group. The region sequenced encodes the last 62 amino-acids of the polymerase and the entire coat protein of the four isolates, plus the 3′ non-translated region of LMV-Gr5 and -Yar. The Greek and Yemenite isolates studied are all very aggressive on lettuce, are able to overcome the resistance genes mo11 and mo12 and belong to the two phylogenetic groups which have so far been only partially characterised. As for other Potyviruses, the core and the C-terminal regions of the coat protein are highly conserved among all isolates whereas the N-terminus is more variable. No amino acid change in the coat protein or carboxy-terminal part of the polymerase could be related to the resistance-breaking properties of the isolates analysed. The sequences obtained provide the basis for the rapid typing of LMV isolates using the restriction pattern of segments of cDNA amplified by PCR.

Natural infection of Alstroemeria caryophyllea with ornithogalum mosaic virus.

During a survey for a European Union-funded project on the viruses of Alstroemeria, an A. caryophyllea plant was found expressing virus-like symptoms, including dark green vein banding, necrotic spots, and flower color breaking. In enzyme-linked immunosorbent assays (ELISA), no positive reaction was obtained with antisera to Alstroemeria mosaic, Alstroemeria carla, Cucumber mosaic, Freesia mosaic, or Tobacco rattle virus. A positive ELISA reaction was obtained with potyvirus-specific monoclonal antibodies (Agdia, Elkhart, IN) and antiserum to Ornithogalum mosaic virus (OrMV) (1). In electron microscopy leaf dip preparations of A. caryophyllea, potyvirus-like particles were observed. Using sapinoculation, the virus was transferred to Chenopodium amaranticolor and C. quinoa, resulting in local lesions 6 days postinoculation. The presence of OrMV in both Chenopodium spp. was confirmed by electron microscopy and ELISA with antiserum to OrMV. Sequence alignment of DNA fragments (740 bp) obtained in immunocapture-reverse transcription-polymerase chain reaction on RNA isolated from the suspect virus, using a potyvirus-specific primer set (2), showed 91% homology with the corresponding region of OrMV RNA (GenBank accession no. D00615). The results confirm the infection of A. caryophyllea by OrMV. This is the first report of natural infection of Alstroemeria by OrMV. References: (1) J. T. Burger and M. B. von Wechmar. Phytopathology 79:385, 1989. (2) R. A. A. van der Vlugt et al. Phytopathology 89:148, 1999.

A bead-based suspension array for the multiplexed detection of begomoviruses and their whitefly vectors

Bead-based suspension array systems enable simultaneous fluorescence-based identification of multiple nucleic acid targets in a single reaction. This study describes the development of a novel approach to plant virus and vector diagnostics, a multiplexed 7-plex array that comprises a hierarchical set of assays for the simultaneous detection of begomoviruses and Bemisia tabaci, from both plant and whitefly samples. The multiplexed array incorporates genus, species and strain-specific assays, offering a unique approach for identifying both known and unknown viruses and B. tabaci species. When tested against a large panel of sequence-characterized begomovirus and whitefly samples, the array was shown to be 100% specific to the homologous target. Additionally, the multiplexed array was highly sensitive, efficiently and concurrently determining both virus and whitefly identity from single viruliferous whitefly samples. The detection limit for one assay within the multiplexed array that specifically detects Tomato yellow leaf curl virus-Israel (TYLCV-IL) was quantified as 200fg of TYLCV-IL DNA, directly equivalent to that of TYLCV-specific qPCR. Highly reproducible results were obtained over multiple tests. The flexible multiplexed array described in this study has great potential for use in plant quarantine, biosecurity and disease management programs worldwide.

Natural infection of Alstroemeria brasiliensis with Lily mottle virus

During a survey for a European Union-funded project on viruses of Alstroemeria, two A. brasiliensis plants were found expressing virus-like symptoms, including leaf chlorosis with deep-green oval spots and flower color breaking. In enzyme-linked immunosorbent assays (ELISA), no positive reaction was obtained with antisera to Alstroemeria mosaic, Alstroemeria carla, Cucumber mosaic, Freesia mosaic, or Tobacco rattle virus or potyvirus-specific monoclonal antibodies (Agdia, Elkhart, IN). ELISA reactions were positive with antisera to Lily mottle (LMoV) and Rembrandt tulip breaking viruses (1). In electron microscopy preparations of A. brasiliensis, potyvirus-like particles were observed. Using sap-inoculation, the virus was transferred to a range of host species. Chenopodium quinoa, Nicotiana occidentalis accession 37B, and N. occidentalis subsp. obliqua (P1) expressed local lesions; N. clevelandii expressed local and systemic mottle; and N. benthamiana expressed local lesions, systemic vein yellowing, and leaf crinkling. Isolated total RNA from infected N. benthamiana was used for initial cDNA synthesis and polymerase chain reaction amplification with a potyvirus-specific primer set (2). The amplicon (≈670 bp) was cloned and sequenced. The sequence showed 92% homology with the corresponding region of LMoV RNA (GenBank accession no. S44147). The results confirm the infection of A. brasiliensis with LMoV. This is the first report of natural infection of Alstroemeria by LMoV. References: (1) E. L. Dekker et al. J. Gen. Virol. 74:881, 1993. (2) R. A. A. van der Vlugt et al. Phytopathology 89:148, 1999.