Geographical distribution, genetic studies and vector transmission of alfalfa enamovirus-1 infecting alfalfa crop in Argentina.

Alfalfa (Medicago sativa L.) growing areas of Argentina were surveyed between 2010 and 2018 to determine the geographical distribution and analyse the genetic diversity among alfalfa enamovirus-1 (AEV-1) isolates. The virus was detected in all 17 surveyed alfalfa-producing provinces, with a prevalence of 64%. The plant virus AEV-1 is widely distributed in the country, and its transmission vector has been unknown until now. Here we show that the black aphid Aphis craccivora can transmit AEV-1. The CP sequence identity among 16 AEV-1 isolates from Argentina was from 98 to 100% and from 98.9 to 100% at nucleotide and amino acid levels, respectively, indicating a low level of sequence variation among these isolates. The phylogenetic analysis based on the complete nucleotide sequence of the CP gene indicated that AEV-1 isolates are closely related and clustered in a monophyletic group. These results suggest that AEV-1 has spread very recently in Argentina. In the present study, we report the geographical distribution of AEV-1 in the main alfalfa-growing areas of Argentina and, for the first time, identify an insect vector and describe the CP gene diversity of an enamovirus worldwide.

Phylodynamics of sunflower chlorotic mottle virus, an emerging pathosystem.

Distribution and epidemiological patterns of sunflower chlorotic mottle virus (SCMoV) in sunflower (Helianthus annuus L.) growing areas in Argentina were studied from 2006 to 2017. The virus was detected exclusively in the Pampas region (Entre Ríos, Santa Fe, Córdoba, La Pampa and Buenos Aires provinces). Phylodynamic analyses performed using the coat protein gene of SCMoV isolates from sunflower and weeds dated the most recent common ancestor (MRCA) back to 1887 (HPD95% = 1572-1971), which coincides with the dates of sunflower introduction in Argentina. The MRCA was located in the south of Buenos Aires province and was associated with sunflower host (posterior probability for the ancestral host, ppah = 0.98). The Bayesian phylodynamic analyses revealed the dispersal patterns of SCMoV, suggesting a link between natural host diversity, crop displacement by human activities and virus spread.

Redefining the medicago sativa alphapartitiviruses genome sequences.

In alfalfa samples analyzed by hightroughput sequencing, four de novo assembled contigs encoding gene products showing identities to alphapartitiviruses proteins were found based on BlastX analysis. The predicted amino acid (aa) sequences of two contigs presented 99-100% identity to the RNA-dependent RNA polymerase (RdRp) and the capsid protein (CP) of the recently reported medicago sativa alphapartitivirus 1 (MsAPV1). In addition, the remaining two contigs shared only 56% (CP) and 70% (RdRp) pairwise aa identity with the proteins of MsAPV1, suggesting that these samples presented also a novel Alphapartitivirus species. Further analyses based on complete genome segments termini and the presence/absence of alphapartitivirus RNA in several samples and public alfalfa RNA datasets corroborated the identification of two different alphapartitivirus members. Our results likely indicate that the reported MsAPV1 genome was previously reconstructed with genome segments of two different alphapartitiviruses. Overall, we not only revisited the MsAPV1 genome sequence but also report a new tentative alphapartitivirus species, which we propose the name medicago sativa alphapartitivirus 2. In addition, the RT-PCR detection of both MsAPV1 and MsAPV2 in several alfalfa cultivars suggests a broad distribution of both viruses.

Genome characterization of an Argentinean isolate of alfalfa leaf curl virus.

We investigated the molecular characteristics of an Argentinean isolate of alfalfa leaf curl virus (ALCV-Arg), a virus of the genus Capulavirus in the family Geminiviridae that was isolated from alfalfa plants showing dwarfism. The genome was found to be 2,750 nucleotides in length. In pairwise comparisons, this ALCV isolate shared 83.2% to 92.6% sequence identity with European ALCV isolates. Sequence comparisons and phylogenetic analysis showed that this isolate combines features of strains A and B of ALCV. Recombination analysis showed that ALCV-Arg is a recombinant isolate that was generated by intraspecific recombination between ALCV strains A and B. The results of this study not only show that ALCV-Arg is unique because it combines features of strains A and B but also show that ALCV naturally infects this forage crop on the American continent.

Complete genome sequence of sunflower ring blotch virus, a new potyvirus infecting sunflower in Argentina.

The complete genome sequence of sunflower ring blotch virus (SuRBV), a previously undescribed potyvirus infecting sunflower in Argentina, is reported. The SuRBV genome comprises 9555 nucleotides (nt) and encodes a polyprotein of 3061 amino acids, flanked by 5' and 3' untranslated regions of 117 and 255 nt, respectively. Phylogenetic analysis showed that SuRBV belongs to the potato virus Y (PVY) subgroup and clusters together with sunflower chlorotic mottle virus and bidens mosaic virus. Percentage nucleotide identity between the whole genomes of SuRBV and BiMV was 70.6%, suggesting SuRBV should be considered a distinct species in the genus Potyvirus.

Analysis of the coding-complete genomic sequence of groundnut ringspot virus suggests a common ancestor with tomato chlorotic spot virus.

Groundnut ringspot virus (GRSV) and tomato chlorotic spot virus (TCSV) share biological and serological properties, so their identification is carried out by molecular methods. Their genomes consist of three segmented RNAs: L, M and S. The finding of a reassortant between these two viruses may complicate correct virus identification and requires the characterization of the complete genome. Therefore, we present for the first time the complete sequences of all the genes encoded by a GRSV isolate. The high level of sequence similarity between GRSV and TCSV (over 90 % identity) observed in the genes and proteins encoded in the M RNA support previous results indicating that these viruses probably have a common ancestor.

Taxonomy of the order Mononegavirales: update 2016.

In 2016, the order Mononegavirales was emended through the addition of two new families (Mymonaviridae and Sunviridae), the elevation of the paramyxoviral subfamily Pneumovirinae to family status (Pneumoviridae), the addition of five free-floating genera (Anphevirus, Arlivirus, Chengtivirus, Crustavirus, and Wastrivirus), and several other changes at the genus and species levels. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).

Complete genome sequence of a new enamovirus from Argentina infecting alfalfa plants showing dwarfism symptoms.

Alfalfa dwarf disease, probably caused by synergistic interactions of mixed virus infections, is a major and emergent disease that threatens alfalfa production in Argentina. Deep sequencing of diseased alfalfa plant samples from the central region of Argentina resulted in the identification of a new virus genome resembling enamoviruses in sequence and genome structure. Phylogenetic analysis suggests that it is a new member of the genus Enamovirus, family Luteoviridae. The virus is tentatively named "alfalfa enamovirus 1" (AEV-1). The availability of the AEV-1 genome sequence will make it possible to assess the genetic variability of this virus and to construct an infectious clone to investigate its role in alfalfa dwarfism disease.

Complete genome sequence and integrated protein localization and interaction map for alfalfa dwarf virus, which combines properties of both cytoplasmic and nuclear plant rhabdoviruses.

We have determined the full-length 14,491-nucleotide genome sequence of a new plant rhabdovirus, alfalfa dwarf virus (ADV). Seven open reading frames (ORFs) were identified in the antigenomic orientation of the negative-sense, single-stranded viral RNA, in the order 3'-N-P-P3-M-G-P6-L-5'. The ORFs are separated by conserved intergenic regions and the genome coding region is flanked by complementary 3' leader and 5' trailer sequences. Phylogenetic analysis of the nucleoprotein amino acid sequence indicated that this alfalfa-infecting rhabdovirus is related to viruses in the genus Cytorhabdovirus. When transiently expressed as GFP fusions in Nicotiana benthamiana leaves, most ADV proteins accumulated in the cell periphery, but unexpectedly P protein was localized exclusively in the nucleus. ADV P protein was shown to have a homotypic, and heterotypic nuclear interactions with N, P3 and M proteins by bimolecular fluorescence complementation. ADV appears unique in that it combines properties of both cytoplasmic and nuclear plant rhabdoviruses.

Complete nucleotide sequence of Alfalfa mosaic virus isolated from alfalfa (Medicago sativa L.) in Argentina.

The complete nucleotide sequence of an Alfalfa mosaic virus (AMV) isolate infecting alfalfa (Medicago sativa L.) in Argentina, AMV-Arg, was determined. The virus genome has the typical organization described for AMV, and comprises 3,643, 2,593, and 2,038 nucleotides for RNA1, 2 and 3, respectively. The whole genome sequence and each encoding region were compared with those of other four isolates that have been completely sequenced from China, Italy, Spain and USA. The nucleotide identity percentages ranged from 95.9 to 99.1 % for the three RNAs and from 93.7 to 99 % for the protein 1 (P1), protein 2 (P2), movement protein and coat protein (CP) encoding regions, whereas the amino acid identity percentages of these proteins ranged from 93.4 to 99.5 %, the lowest value corresponding to P2. CP sequences of AMV-Arg were compared with those of other 25 available isolates, and the phylogenetic analysis based on the CP gene was carried out. The highest percentage of nucleotide sequence identity of the CP gene was 98.3 % with a Chinese isolate and 98.6 % at the amino acid level with four isolates, two from Italy, one from Brazil and the remaining one from China. The phylogenetic analysis showed that AMV-Arg is closely related to subgroup I of AMV isolates. To our knowledge, this is the first report of a complete nucleotide sequence of AMV from South America and the first worldwide report of complete nucleotide sequence of AMV isolated from alfalfa as natural host.

Redox-related metabolites and gene expression modulated by sugar in sunflower leaves: similarities with Sunflower chlorotic mottle virus-induced symptom.

Sugars are part of an integrated redox system, since they are key regulators of respiration and photosynthesis, and therefore of the levels of reducing power, ATP and ROS. These elements are major determinants of the cellular redox state, which is involved in the perception and regulation of many endogenous and environmental stimuli. Our previous findings suggested that early sugar increase produced during compatible Sunflower chlorotic mottle virus (SuCMoV) infection might modulate chlorotic symptom development through redox state alteration in sunflower. The purpose of this work was to characterize redox-related metabolites and gene expression changes associated with high sugar availability and symptom development induced by SuCMoV. The results show that sugar caused an increase in glutathione, ascorbate, pyridine nucleotides, and ATP. In addition, higher sugar availability reduced hydrogen peroxide and ΦPSII. This finding suggests that high sugar availability would be associated with cellular redox alteration and photoinhibitory process. The expression of the genes analyzed was also strongly affected by sugar, such as the down-regulation of psbA and up-regulation of psbO and cp29. The expression level of cytoplasmic (apx-1 and gr)- and chloroplastic (Fe-sod)-targeted genes was also significantly enhanced in sugar-treated leaves. Therefore, all these responses suggest that sugars induce chloroplastic redox state alteration with photoinhibition process that could be contributing to chlorotic symptom development during SuCMoV infection.

The chlorotic symptom induced by Sunflower chlorotic mottle virus is associated with changes in redox-related gene expression and metabolites.

Systemic infections are commonly associated with changes in host metabolism and gene expression. Sunflower chlorotic mottle virus (SuCMoV) causes systemic infection with sugar increase, photoinhibition and increase in antioxidant enzyme activities before chlorotic symptom appearance in sunflower leaves. The aim of this study was to determine if chlorotic symptom development induced by SuCMoV infection is accompanied by changes in different redox-related metabolites and transcripts. Symptom development was analyzed in the second pair of leaves (systemic infection) at different post-inoculation times: before symptom appearance (BS, 4 dpi), and at an early (ES, 7 dpi) and later stage (LS, 12 dpi) of symptom expression. The results showed that the virus reaches the second pair of leaves at 4 dpi. A positive correlation between chlorotic symptom and number of viral copies was also observed. Changes in hydrogen peroxide, glutathione, pyridine nucleotides and ATP content were observed since symptom appearance (ES, 7 dpi). The expression of some of the genes analyzed was also strongly affected by SuCMoV infection. Specifically, down-regulation of both chloroplast-encoded genes and chloroplast-targeted genes: psbA, rbcS, Cu/Zn sod, Fe sod, phosphoglycolate phosphatase, psbO, psaH and fnr was present, whereas the expression of cytoplasmic-targeted genes, apx1, and Cu/Zn sod was up-regulated. Mitochondrial Mn sod decreased at BS stage and aox decreased only at ES stage. Peroxisomal catalase (cat-2) was lower at BS and LS stages. All these results suggest that SuCMoV infection induces progressive changes in determinants of redox homeostasis associated with chlorotic symptom development.

Are Sunflower chlorotic mottle virus infection symptoms modulated by early increases in leaf sugar concentration?

Symptom development in a susceptible sunflower line inoculated with Sunflower chlorotic mottle virus (SuCMoV) was followed in the second pair of leaves at different post-inoculation times: before symptom expression (BS), at early (ES) and late (LS) symptom expression. Sugar and starch increases and photoinhibition were observed as early effects BS, and were maintained or enhanced later on, however, chlorophyll loss was detected only at LS. Photoinhibition correlated with a drastic decrease in D1 protein level. The progress of infection was accompanied by decreasing levels of apoplastic reactive oxygen species (ROS). In infected leaves, higher antioxidant enzyme activities (superoxide dismutase, SOD; ascorbate peroxidase, APX; glutathione reductase, GR) were observed from BS. The purpose of this work was to evaluate whether the early increases in carbohydrate accumulation may participate in SuCMoV symptom expression. Similar effects on photoinhibition, apoplastic ROS generation and antioxidant activity were generated when healthy leaves were treated with sugars. These results suggest that photoinhibitory processes and lower apoplastic superoxide levels induced by SuCMoV infection may be modulated by sugar increases.

Natural infection of Viola cornuta (Violaceae) with Cucumber mosaic virus, subgroup I.

Plants of Viola cornuta displaying typical virus symptoms were observed during spring 2003 in a plant nursery in Córdoba, central Argentina. Electron microscopic examinations of symptomatic leaf samples revealed the presence of isometric virus-like particles about 30 nm in diameter. Subsequent serological analysis allowed the identification of the pathogen as a subgroup I strain of Cucumber mosaic virus (CMV). These results were confirmed by antigen capture--reverse transcription--polymerase chain reaction with specific CMV primers, and digestion with a restriction enzyme. This is the first report of CMV infecting V. cornuta in Argentina.

Natural infection of Viola cornuta (Violaceae) with Cucumber mosaic virus, subgroup I

Plants of Viola cornuta displaying typical virus symptoms were observed during spring 2003 in a plant nursery in Córdoba, central Argentina. Electron microscopic examinations of symptomatic leaf samples revealed the presence of isometric virus-like particles about 30 nm in diameter. Subsequent serological analysis allowed the identification of the pathogen as a subgroup 1 strain of Cucumber mosaic virus (CMV). These results were confirmed by antigen capture - reverse transcription - polymerase chain reaction with specific CMV primers, and digestion with a restriction enzyme. This is the first report of CMV infecting V cornuta in Argentina