Sequencing of two sunflower chlorotic mottle virus isolates obtained from different natural hosts shed light on its evolutionary history.

Sunflower chlorotic mottle virus (SuCMoV), the most prevalent virus of sunflower in Argentina, was reported naturally infecting not only sunflower but also weeds. To understand SuCMoV evolution and improve the knowledge on its variability, the complete genomic sequences of two SuCMoV isolates collected from Dipsacus fullonum (-dip) and Ibicella lutea (-ibi) were determined from three overlapping cDNA clones and subjected to phylogenetic and recombination analyses. SuCMoV-dip and -ibi genomes were 9,953-nucleotides (nt) long; their sequences contained an open reading frame of 9,561 nucleotides, which encoded a polyprotein of 3,187 amino acids flanked by a 5'-noncoding region (NCR) of 135 nt and a 3'-NCR of 257 nt. SuCMoV-dip and -ibi genome nucleotide sequences were 90.9 identical and displayed 90 and 94.6 % identity to that of SuCMoV-C, and 90.8 and 91.4 % identity to that of SuCMoV-CRS, respectively. P1 of SuCMoV-dip and -ibi was 3-nt longer than that of SuCMoV-CRS, but 12-nt shorter than that of SuCMoV-C. Two recombination events were detected in SuCMoV genome and the analysis of d(N)/d(S) ratio among SuCMoV complete sequences showed that the genomic regions are under different evolutionary constraints, suggesting that SuCMoV evolution would be conservative. Our findings provide evidence that mutation and recombination would have played important roles in the evolutionary history of SuCMoV.

Development of a full-length infectious clone of sunflower chlorotic mottle virus (SuCMoV).

A full-length cDNA clone (p35SuCMoV) of the sunflower chlorotic mottle virus common strain (SuCMoV-C) genomic RNA was constructed. Three cDNA fragments covering the whole genome of SuCMoV-C were cloned between a cauliflower mosaic virus 35S promoter and a nopaline synthase terminator. Mechanical inoculation of sunflower and Nicotiana occidentalis seedlings with p35SuCMoV DNA led to systemic infection. Symptoms induced by p35SuCMoV were similar to those caused by the wild-type SuCMoV-C but appeared four days later. Infection was confirmed by a western blot test, electron microscopy, RT-PCR and inoculation of progeny virions to sunflower seedlings. This is the first report about the construction of a biologically active, full-length cDNA copy of the SuCMoV-C RNA genome.

First Report of a Rhabdovirus Infecting Alfalfa in Argentina.

Alfalfa (Medicago sativa L.) is a major forage crop in Argentina with an estimated cultivated area of 4 million ha in the 2009-2010 season, which constitutes a primary component for the animal production chain. In early summer of 2010, alfalfa plants showing virus-like symptoms were identified in 20 commercial fields in La Pampa Province with 95% disease prevalence. Diseased plants had shortened internodes, a bushy appearance, deformations, puckering, epinasty of leaflet blades, vein enations, and varying sized papillae on the adaxial leaflet surfaces. Similar symptoms were observed in alfalfa crops in Buenos Aires, Cordoba, Santa Fe, and Santiago del Estero provinces. Electron microscopy (EM) and molecular assays were performed on leaf tissue from one asymptomatic and two symptomatic plants. EM of ultrathin sections revealed membrane-bound bullet-shaped particles associated with the endoplasmic reticulum of phloem cells from symptomatic plants only. Total RNA was extracted from symptomatic and asymptomatic plants with the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) for a template in one-step reverse transcription (RT)-PCR assays with the Access RT-PCR Kit (Promega, Madison, WI). RT-PCR assays employed degenerate primers targeting conserved regions of plant rhabdovirus polymerase (L) genes (2). An amplicon of approximately 1 kilobase pairs (detected only from symptomatic plants) was gel purified with the Wizard SV Gel and PCR Clean-Up System (Promega), cloned into pGEM-T Easy Vector System (Promega), and sequenced. Three independents clones were sequenced in both directions at Macrogen Inc. (Korea Republic) to generate a consensus sequence (GenBank Accession No. HQ380230) and compared to sequences of other plant rhabdoviruses available on GenBank. The partial L gene sequence of the alfalfa-infecting rhabdovirus shared highest nucleotide (68.0%) and amino acid (76.5%) sequence identity with the cytorhabdovirus Strawberry crinkle virus (Accession No. AY331390). A phylogenetic tree based on partial amino acid sequences of the polymerase gene indicated the alfalfa-infecting virus was more closely related to cytorhabdoviruses than to nucleorhabdoviruses. Symptoms observed resembled those reported for alfalfa plants infected with a plant rhabdovirus named Alfalfa enation virus (1), which has never been fully characterized. Collectively, the data implicate the observed rhabdovirus as the etiological agent. To our knowledge, this is the first report in Argentina (and South America) of a rhabdovirus infecting alfalfa. Additional field surveys and monitoring of vector/s and yield losses need to be conducted. References: (1) B. Alliot and P. A. Signoret. Phytopathol. Z. 74:69, 1972. (2) R. L. Lamprecht et al. Eur. J. Plant Pathol. 123:105, 2009.

Molecular characterization of Sunflower chlorotic mottle virus: a member of a distinct species in the genus Potyvirus.

The complete nucleotide (nt) and deduced amino acid (aa) sequences of the C (common) and CRS (chlorotic ringspot) Argentine strains of SuCMoV have been determined. The SuCMoV-C RNA genome consists of 9,965 nt, whereas indels within the P1 coding region of SuCMoV-CRS make its genomic length 15 nt shorter. Nucleotide and aa sequence identities between the polyproteins of the C and CRS strains of SuCMoV were 92.3 and 95.6%, respectively. Pairwise comparisons between the polyproteins of the C and CRS strains of SuCMoV and the viruses of the Potato virus Y (PVY) subgroup revealed identities of 66.5-66.9% at the nt level and 69.7-69.8% at the aa level. These results and phylogenetic analyses show that although SuCMoV strains cluster together with the potyviruses belonging to the PVY subgroup, SuCMoV should be considered a member of a distinct species in the genus Potyvirus.

Occurrence, Prevalence, and Distribution of Viruses Infecting Peanut in Argentina.

This is the first survey to determine the occurrence, prevalence, and distribution of peanut (Arachis hypogaea) viral diseases in Argentina. It was conducted in the province of Córdoba, which has 92% of the country's peanut production. It included the main peanut viruses Peanut mottle virus (PeMoV), Peanut stripe virus (PStV), Cucumber mosaic virus (CMV), Peanut stunt virus (PSV), Tomato spotted wilt virus (TSWV), and Groundnut ringspot virus (GRSV). Leaf samples from 1,028 individual peanut plants with virus-like symptoms and 986 samples from asymptomatic plants were collected in six counties of Córdoba over 3 years and serologically tested for the presence of viruses. PeMoV was the most frequently detected virus, found in 58.8, 34.2, and 23.4% of samples from the 2003-04, 2004-05, and 2005-06 growing seasons, respectively, and it was found in all sampled counties. Also, it was the only virus detected in asymptomatic plants. Less than 4% of symptomatic plants were infected with CMV or GRSV; 0.5, 3.6, and 2% of samples were positive for CMV; and 0.5, 3.1, and 1.6% were positive for GRSV in the 2003-04, 2004-05 and 2005-06 seasons, respectively. Some mixed infections were found: CMV-PeMoV and GRSV-PeMoV. During this survey, PSV, PStV, and TSWV were not detected in any peanut samples.

Sequencing of the bicistronic genome segments S7 and S9 of Mal de Río Cuarto virus (Fijivirus, Reoviridae) completes the genome of this virus.

The nucleotide sequences of genomic segments S7 and S9 of Mal de Río Cuarto virus (MRCV, Fijivirus group II) have been determined, thus completing the entire genome sequence of the virus. These segments showed a non-overlapping bicistronic structure, as in other members of the genus. MRCV S7 ORF-1 had a length of 1086 bp and encoded a 41.5 kDa putative polypeptide, whereas MRCV S7 ORF-2 had a length of 930 bp and encoded a 36.8 kDa putative polypeptide. Proteins of 39 and 20.5 kDa were predicted for the 1014 bp long MRCV S9 ORF-1 and the 537 bp long MRCV S9 ORF-2, respectively. The terminal 5' and 3' sequences of both segments were 5'AAGUUUUU3' and 5'CAGCUnnnGUC3', respectively. Specific imperfect inverted repeats of each segment were identified. Comparison of the predicted proteins with those of related virus genome segments counterparts in maize rough dwarf virus (MRDV) and rice black streaked dwarf virus (RBSDV), showed 64.5-44.3% identities. These values are lower than those resulting from comparisons between MRDV and RBSDV. The topology of the trees obtained using the complete nucleotide and amino acid sequences of MRCV S7 and MRCV S9 was consistent with the analysis of the other MRCV segments previously published.

Occurrence of a Reovirus Infecting Maize in India.

During the 1998-99 season, maize plants showing viruslike symptoms were observed at two locations in the Andhra Pradesh state in Southern India. Several hybrids were evaluated at Hyderabad in a replicated yield trial and most were affected, with disease incidence ranging from 4.4 to 61.8% of the plants among plots. Hybrid 4642 (Proagro 3-way hybrid, late maturity) and the pre-commercial hybrid YLY102 were the most susceptible, whereas the popular hybrid 4640 was among the least susceptible entries. In seed production fields near Eluru, incidence ranged from 10 to 15% among plots, with the female parent of hybrid 4210 (Proagro 3-way hybrid, early maturity) being especially affected. Symptoms observed in hybrids varied, presumably, according to the infection time and included severe plant dwarfing, dark-green leaves, enations on the lower leaf surface, and small malformed ears with few or no kernels. Symptomatic and asymptomatic field plants (root and leaf tissues) were tested by ds-RNA polyacrylamide gel electrophoresis and by double-antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA) with antiserum to Mal de río cuarto virus (MRCV), a Reoviridae-Fijivirus member. MRCV and Maize rough dwarf virus (MRDV) were selected as controls because the symptoms were similar to those caused by these maize viruses (1,2). ds-RNA gels from symptomatic plants showed 10 bands with banding patterns different from those of MRCV or MRDV. DAS-ELISA indicated a distant relationship to MRCV. These results provide evidence of a reovirus infection to maize hybrids in India and suggest that a virus belonging to the family Reoviridae, genus Fijivirus is causing this new disease. The high disease incidence and the severity of symptoms should alert breeders and pathologists in southern Asia about potential yield losses. References: (1) G. Boccardo and R. G. Milne. 1984. Descriptions of Plant Viruses 294. Inst. Hortic. Res., Wellesbourne. (2) C. Marzachi et al. J. Plant Dis. Prot. 106:431-436, 1999.

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.

Recent Outbreak of "Mal de Rio Cuarto" Virus on Corn in Argentina.

"Mal de Río Cuarto" (MRC) is the most important viral disease affecting corn in Argentina. Reovirus-like particles were observed in diseased plants (1,4) and were later serologically related to an isolate of maize rough dwarf virus (3), though this relationship was recently questioned (2). Based on estimates of the prevalence and severity of MRC and yield losses, government agencies, corn hybrid seed companies, and growers agreed that the worst epidemic in the country occurred during the 1996 to 1997 agricultural year. Approximately 300,000 ha of corn were affected by the disease and yield losses were estimated at $120 million. Affected areas included the central and southern Santa Fe, the central, northern, southeastern, and western Buenos Aires, and the eastern and southern (originally the endemic center of MRC in Río Cuarto County) parts of Córdoba. Virus infections were confirmed by double-antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) in root samples from each surveyed location, using an antiserum to MRC virus. The occurrence of MRC in non-endemic areas suggests an unusual phenological coincidence of high vector populations, abundant natural virus reservoirs, and susceptible stages in the crop. Most commercial hybrids surveyed were apparently susceptible to the virus, although some were tolerant. References: (1) O. E. Bradfute et al. Phytopathology 71:205, 1981. (2) C. Marzachi et al. Sem. Virol. 6:103, 1995. (3) R. G. Milne et al. Phytopathology 73:1290, 1983. (4) S. F. Nome et al. Phytopathol. Z. 101:7, 1981.

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.