First Report of Mosaic Disease Caused by Colombian datura virus on Solanum lycopersicum Plants Commercially Cultivated in Japan.

In 2009, some commercially grown tomato (Solanum lycopersicum) plants in Chiba Prefecture, Japan, exhibited mosaic symptoms. Ten plants from a total of about 72,000 cultivated plants in the greenhouses showed such symptoms. To identify the causal agent, sap from leaves of the diseased plants was inoculated into Chenopodium quinoa and Nicotiana benthamiana plants. Local necrotic lesions appeared on inoculated leaves of C. quinoa, but no systemic infection was observed. Systemic mosaic symptoms were observed on the N. benthamiana plants inoculated. Single local lesion isolation was performed three times using C. quinoa to obtain a reference isolate for further characterization. N. benthamiana was used for propagation of the isolate. Sap from infected leaves of N. benthamiana was mechanically inoculated into three individual S. lycopersicum cv. Momotaro. Symptoms appearing on inoculated tomatoes were indistinguishable from those of diseased tomato plants found initially in the greenhouse. Flexuous, filamentous particles, ~750 nm long, were observed by electron microscopy in the sap of the tomato plants inoculated with the isolate, indicating that the infecting virus may belong to the family Potyviridae. To determine genomic sequence of the virus, RT-PCR was performed. Total RNA was extracted from the tomato leaves experimentally infected with the isolate using an RNeasy Plant Mini kit (QIAGEN, Hilden, Germany). RT-PCR was performed by using a set of universal, degenerate primers for Potyviruses as previously reported (2). Amplicons (~1,500 bp) generated by RT-PCR were extracted from the gels using the QIAquick Gel Extraction kit (QIAGEN) and cloned into pCR-BluntII TOPO (Invitrogen, San Diego, CA). DNA sequences of three individual clones were determined using a combination of plasmid and virus-specific primers, showing that identity among three clones was 99.8%. A consensus nucleotide sequence of the isolate was deposited in GenBank (AB823816). BLASTn analysis of the nucleotide sequence determined showed 99% identity with a partial sequence in the NIb/coat protein (CP) region of Colombian datura virus (CDV) tobacco isolate (JQ801448). Comparison of the amino acid sequence predicted for the CP with previously reported sequences for CDV (AY621656, AJ237923, EU571230, AM113759, AM113754, and AM113761) showed 97 to 100% identity range. Subsequently, CDV infection in both the original and experimentally inoculated plants was confirmed by RT-PCR using CDV-specific primers (CDVv and CDVvc; [1]), and, hence, the causal agent of the tomato disease observed in greenhouse tomatoes was proved to be CDV. The first case of CDV on tomato was reported in Netherlands (3), indicating that CDV was transmitted by aphids from CDV-infected Brugmansia plants cultivated in the same greenhouse. We carefully investigated whether Brugmansia plants naturally grew around the greenhouses, but we could not find them inside or in proximity to the greenhouses. Therefore, sources of CDV inoculum in Japan are still unclear. This is the first report of a mosaic disease caused by CDV on commercially cultivated S. lycopersicum in Japan. References: (1) D. O. Chellemi et al. Plant Dis. 95:755, 2011. (2) J. Chen et al. Arch. Virol. 146:757, 2001. (3) J. Th. J. Verhoeven et al. Eur. J. Plant. Pathol. 102:895, 1996.

Characterization of a Potyvirus Infecting Sunflower in Argentina.

A virus causing chlorotic mottling symptoms on sunflower was found in various locations in Argentina. Symptoms were small chlorotic spots, yellow blotches on leaves, and plant stunting. Virus transmission efficiency by mechanical inoculation was 73 to 100%, and by Myzus persicae was 31 to 49%. The host range included members of the Amaranthaceae, Asteraceae, Chenopodiaceae, and Solanaceae families. Electron microscopy of leaf dips from infected plants revealed flexuous particles 17 nm wide and 770 nm long. Cytoplasmic laminar aggregates and pinwheel inclusions were observed in ultrathin sections. Purified virus preparations analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved a capsid protein of 33 kDa. A monoclonal antibody to aphid-transmitted potyviruses reacted with the capsid protein of this virus. In dot blot immunoassays, a polyclonal antiserum (early bleeding) reacted with infected sunflowers and weakly with Bidens mottle potyvirus, but not with either maize dwarf mosaic potyvirus or potato virus Y. The evidence suggests that a potyvirus is infecting sunflower, and a partial characterization of the causal agent is reported.

Nucleotide sequence at the 3'-terminal region of sweet potato feathery mottle virus (ordinary strain, SPFMV-O) RNA.

cDNA encoding the 3'-terminal region of sweet potato feathery mottle virus (ordinary strain, SPFMV-O) RNA was cloned and sequenced. The 2324-bp cDNA contained an open reading frame (ORF) of 2066 bp followed by an 3' non-coding region and a poly(A) region. The ORF covered the coat protein and the carboxy terminus of the nuclear inclusion 'b' protein (NIb).

Molecular characterization of a new potyvirus infecting sunflower.

We have sequenced 1873 nucleotides from the 3'-end region of a sunflower potyvirus genome including the 3'-NIb protein coding region (813 nucleotides), the entire coat protein coding region (807 nucleotides) and 3'-NCR (253 nucleotides), excluding the poly (A) tail. Amino acids identity of the whole CP between the sunflower virus and Potyvirus members ranged from 49.5% (SCMV) to 81.5% (PVY-NsNr), and the core ranged from 55% (TVMV) to 87% (PVY-NsNr; PepMoV). The 3'-NCR nucleotides showed 38.7% homology to PeSMV and 61% to PepMoV-C. The sequence of 3' end region and analysis of phylogenetic relationships suggest this sunflower virus could belong to PVY subgroup and the name of "sunflower chlorotic mottle virus" (SuCMoV) is proposed. This is the first report on the partial nucleotide sequence of a potyvirus infecting sunflower.

Nucleotide sequence analysis of two nuclear inclusion body and coat protein genes of a sweet potato feathery mottle virus severe strain (SPFMV-S) genomic RNA.

Recombinant DNA molecules containing cDNA of a sweet potato feathery mottle virus severe strain (SPFMV-S) RNA genome were constructed and the partial nucleotide sequences were determined for three DNA inserts, which cover 4-2 kb from the 3'-terminus excluding the poly (A) tail. This region of the genome consists of an open reading frame of 1340 amino acids (a.a.) and a 3'-non-translated region of 224 nucleotides. The protein products expected were 6K2 (53 a.a.), NIa (435 a.a.), NIb (521 a.a.) and CP (315 a.a.). Among NIa, NIb and coat proteins, the NIb protein was found to be the most conserved (59-68%) when compared to the corresponding proteins of other distinct potyviruses.

Complete nucleotide sequence and genome organization of sweet potato feathery mottle virus (S strain) genomic RNA: the large coding region of the P1 gene.

The complete nucleotide sequence of a sweet potato feathery mottle virus severe strain (SPFMV-S) genomic RNA was determined from overlapping cDNA clones and by directly sequencing viral RNA. The viral RNA genome is 10,820 nucleotides long, excluding the poly(A) tail and contains one open reading frame (ORF) starting at nucleotide 118 and ending at 10,599, potentially encoding a polyprotein of 3,493 amino acids (Mr 393,800). The ORF was followed by a 3' untranslated region of 221 nucleotides. The deduced polyprotein includes P1 (74K), HC-Pro (52K), P3 (46K), 6K1, CI (72K), 6K2, NIa-VPg (22K), NIa-Pro (28K), NIb (60K) and coat (35K) proteins, after an analysis of protein cleavage sites analogous to other potyvirus polyproteins. The polyprotein had a high level of amino acid identity with those of other potyviruses, except in the regions of P1 and P3. The P1 of SPFMV-S RNA has 664 amino acid residues, and is the largest and least similar to those of other potyviruses. HC-Pro and CI show high identity with those of other potyviruses. P3 has relatively low identity, however, the length of P3 was within the range of variability among other potyviruses. The 6K1 protein between P3 and C1 is also highly similar to those of other potyviruses. This is the first report on the complete nucleotide sequence of the sweet potato-infecting virus.

Nucleotide sequence of the capsid protein gene of papaya leaf-distortion mosaic potyvirus.

The DNA complementary to the 3'-terminal 1 404 nucleotides [excluding the poly(A) tail] of papaya leaf-distortion mosaic potyvirus (PLDMV) RNA was cloned and sequenced. The sequence starts within a long open reading frame (ORF) of 1 195 nucleotides and is followed by a 3' non-coding region of 209 nucleotides. Capsid protein (CP) is encoded at the 3' terminus of the ORF. The CP contains 293 residues and has a Mr of 33 277. The CP of PLDMV exhibits 49 to 59% sequence similarity at the amino acid level to the CPs of papaya ringspot potyvirus (PRSV) and other potyviruses. This result is consistent with the absence of a serological relationship between PLDMV and PRSV or other potyviruses. The results support the assignment of PLDMV as a distinct member of the genus Potyvirus.