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.