Sunflower Chlorotic Mottle Virus Research Articles

First report of sunflower chlorotic mottle virus infecting sunflower plants in Brazil

Sunflower (Helianthus annuus) plants showing symptoms of chlorosis, mosaic, chlorotic ringspot, and necrosis on younger leaves were found in a small experimental plot in Piracicaba, in the state of São Paulo, Brazil. Preliminary examinations by transmission electron microscopy of symptomatic leaf tissue revealed flexuous filamentous particles 13-15 nm wide and 700-750 nm long, and cytoplasmatic cylindrical inclusions typical of those found in plant cells infected by members of the Potyvirus genus. Total RNA extracted from symptomatic leaves and subjected to RT-PCR followed by partial nucleotide sequencing confirmed the presence of a potyvirus in the affected plants, which was identified as sunflower chlorotic mottle virus (SuCMoV), a member of the Sunflower chlorotic mottle virus (genus Potyvirus, family Potyviridae) species. Mechanical transmission assays with extracts of symptomatic sunflower leaves reproduced the original symptoms in sunflowers, mosaic symptoms in Zinnia elegans, and chlorotic local lesions in Chenopodium amaranticolor and C. quinoa. Sunflower and zinnia plants became infected after aphid transmission experiments with Myzus persicae. RT-PCR tests using specific primers for SuCMoV confirmed the presence of this virus in experimentally infected plants, meeting the criteria of Koch’s postulate. This is the first report of SuCMoV infecting sunflower plants in Brazil.

The Quest to Identify a New Virus DiseaseofSunflower from Nebraska.

Between 2010 and 2018, sunflower plants exhibiting virus-like symptoms, including stunting, mottling, and chlorotic ringspots on leaves, were observed from commercial fields and research plots from four sites within three distinct counties of western Nebraska (Box Butte, Kimball, and Scotts Bluff). Near identical symptoms from field samples were reproduced on seedlings mechanically in the greenhouse on multiple occasions, confirming the presence of a sap-transmissible virus from each site. Symptomatic greenhouse-inoculated plants from the 2010 and 2011 Box Butte samples tested negative for sunflower mosaic virus (SuMV), sunflower chlorotic mottle virus (SuCMoV), and all potyviruses in general by ELISA and RT-PCR. Similar viral-like symptoms were later observed on plants in a commercial sunflower field in Kimball County in 2014, and again from volunteers in research plots in Scotts Bluff County in 2018. Samples from both of these years were again successfully reproduced on seedlings in the greenhouse as before following mechanical transmissions. Symptom expression for all years began 12 to 14 days after inoculation as mild yellow spots followed by the formation of chlorotic ringspots from the mottled pattern. The culture from 2014 tested negatively for three groups of nepoviruses via RT-PCR, ruling this group out. However, transmission electron microscopy assays of greenhouse-infected plants from both 2014 and 2018 revealed the presence of distinct, polyhedral virus particles. With the use of high throughput sequencing and RT-PCR, it was confirmed that the infections from both years were caused by a new virus in the tombusvirus genus and was proposed to be called Sunflower ring spot mottle virus (SuRSMV). Although the major objective of this project was to identify the causal agent of the disease, it became evident that the diagnostic journey itself, with all the barriers encountered on the 10-year trek, was actually more important and impactful than identification.

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.

Molecular characterization and complete genome of alstroemeria mosaic virus (AlMV).

Even though alstroemeria mosaic virus (AlMV) is one of the most important viruses affecting alstroemeria plants, its genome is only partially available in public sequence databases. High throughput sequencing (HTS) of RNA from alstroemeria plants with symptoms of mosaic and streaking, collected in Lasso-Ecuador, indicated the presence of AlMV and lily symptomless virus. In this study, we aimed to assemble and characterize the complete genome sequence of AlMV. Reads from Illumina sequencing of ribosomal RNA-depleted total RNA were assembled into contigs that were mapped to the sunflower chlorotic mottle virus genome, revealing the 9774 [corrected]bp complete genome sequence of AlMV. Multiple sequence alignment of the AlMV polyprotein with close homologs allowed the identification of ten mature proteins P1, HC-Pro, P3, 6K1, CI, 6K2, NIa-VPg, NIa-Pro, NIb and CP. Furthermore, several potyvirus motifs were identified in the AlMV polyprotein including those related to potyvirus aphid transmission 334KMTC337, 592PTK594 and 2800DAG2802. Phylogenetic analysis based in the polyprotein showed that AlMV belongs to the potato virus Y clade and its closest relative is sunflower ring blotch virus. This study describes the first complete genome of AlMV and its placement within the genus Potyvirus, providing valuable information for future studies on this economically important virus.

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.

Biological and molecular characterisation of Bidens mosaic virus supports its assignment as a member of a distinct species in the genus Potyvirus

The complete nucleotide (nt) sequence of Bidens mosaic virus (BiMV) isolate SP01 was determined and shown to consist of 9,557 nt. Since it shared highest identities in the nt sequence of the whole genome (66-73 %) and in the aa sequence of the polyprotein (60-76 %) with viruses of the potato virus Y subgroup, it was compared with them genetically and biologically. Phylogenetic analysis showed that the closest relative of BiMV is sunflower chlorotic mottle virus, from which it, however, differed significantly in various respects. These results indicate that BiMV should represent a distinct species in the genus Potyvirus.

Sequence variation and protein structure of pipo gene in Potato virus Y

The objectives of this study were to understand the sequence variation and the putative protein structure of pipo gene in the Potato virus Y (PVY) collected from Solanum tuberosum. The pipo gene in PVY was cloned using a pair of degenerate primers designed from its conserved region and its sequences were used to re-construct phylogenetic tree in Potyvirus genera by a Bayesian inference method. An expected fragment of 235 bp was amplified in all 20 samples by RT-PCR and the pipo genes in the 20 samples assayed shared more than 92% nucleotide sequence similarity with the published sequences of PVY strains. Among the 20 pipo gene sequences, 13 polymorphic sites were detected, including 4 parsimony informative sites and 9 singleton variable sites. These results indicate that PVY pipo gene is highly conserved but some sequence variations exist. Further analyses suggest that the pipo gene encodes a hydrophilic protein without signal peptide and transmembrane region. The protein has theoretical isoelectric points (pI) ranging from 11.26 to 11.62 and contains three highly conserved regions, especially between aa 10 and 59. The protein is likely located in the mitochondria and has a-helix secondary structure. Bayesian inference of phylogenetic trees reveals that PVY isolates are clustered in the same branch with high posterior probability, while Sunflower chlorotic mottle virus (SoCMoV) and Pepper severe mosaic virus (PepSMV) are closely related, consisting with the classification of Potyvirus genera using other approaches. Our analyses suggest that the pipo gene can be a new marker for phylogenetic analysis of the genera. The results reported in this paper provide useful insights in the genetic variation and the evolution of PVY and can stimulate further research on structure and function of the PIPO protein.

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.

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.

Complete genome sequence of arracacha mottle virus

Arracacha mottle virus (AMoV) is the only potyvirus reported to infect arracacha (Arracacia xanthorrhiza) in Brazil. Here, the complete genome sequence of an isolate of AMoV was determined to be 9,630 nucleotides in length, excluding the 3' poly-A tail, and encoding a polyprotein of 3,135 amino acids and a putative P3N-PIPO protein. Its genomic organization is typical of a member of the genus Potyvirus, containing all conserved motifs. Its full genome sequence shared 56.2 % nucleotide identity with sunflower chlorotic mottle virus and verbena virus Y, the most closely related viruses.

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.

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, 4dpi), and at an early (ES, 7dpi) and later stage (LS, 12dpi) of symptom expression. The results showed that the virus reaches the second pair of leaves at 4dpi. 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, 7dpi). 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.

Identification and Characterization of a New Strain of Sunflower chlorotic mottle virus, a Potyvirus Infecting Asteraceae in Argentina

AbstractSunflower (Helianthus annuus L.) plants with chlorotic ringspot symptoms were observed during the 2005–2006 crop season in the Southeast of the province of Buenos Aires, Argentina. Symptomatic plants were tested by biological, serological and molecular assays, and the virus isolated was identified as a potyvirus closely related to Sunflower chlorotic mottle virus, common isolate (SuCMoV‐C), the most prevalent virus in sunflower crops in the country. Infected plants were serologically positive when probed with a SuCMoV‐C antiserum. In the 3′‐terminus region, 1304 nucleotides (nt) were sequenced, and it includes the C‐terminal region of the nuclear inclusion b protein (NIb) gene (240 nt), the whole capsid protein (CP) gene (807 nt) and a 3′‐non‐coding region (3′‐NCR) with 257 nt excluding the poly (A) tail. The CP of the Sunflower potyvirus causing chlorotic ringspot (CRS) shared 94.8% aa identity with SuCMoV‐C and 89.2% with SuCMoV‐Zi. The 3′‐NCR shared 94.2% nt sequence identity with SuCMoV‐C. A RT‐PCR/RFLP assay with PvuII and EcoRV restriction enzymes successfully differentiated SuCMoV‐C and the virus isolate causing CRS symptoms. This potyvirus was identified as a new SuCMoV strain, provisionally designated SuCMoV‐CRS.

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.

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.

The argentine wild Helianthus annuus L. genetic resource

Wild Helianthus annuus naturalized in Argentina could be a valuable germplasm source for cultivated sunflower. Five wild populations collected in different environments and with different phenotype expression were evaluated as a genetic resource in a common garden study. The populations showed normal meiosis and produced a good seed set when their plants pollinated the male-sterile inbred line A09 (cms PET1). The wild populations restored more than 80% fertility of the HA89 (cms PET1) and A10 (cms PET1) inbred lines in the F1 generation. The fertility of one male sterile source from Mendoza was restored (>95%) by the maintainer line B10. The fatty acid composition differentiated a population from Entre Rios with a high saturated fatty acid content (>107 g kg-1). Another population from La Pampa showed a high level (>50%) of resistance to the Sunflower Chlorotic Mottle Virus (SuCMoV). No wild populations showed tolerance to imazaphyr sprayed at 2× doses (×=80 g i.a. ha-1). A population collected in the coolest environment showed high tolerance to low temperature (15/5°C, neutral day) in the initial grow stages ( 80%) under water stress (-0.4 MPa) imposed by polyethylene glycol 6000. This population and a second one from southern Buenos Aires showed the lowest leaf temperature increase (<10%) and the highest foliar specific density under artificial drought during the R4 to R6 reproductive stages. It was concluded that wild H. annuus naturalized in central Argentina can supply some useful traits for sunflower breeding.

Dipsacus fullonum: an Alternative Host of Sunflower chlorotic mottle virus in Argentina

Abstract Dipsacus fullonum L. is a biennial weed that is widely spread in the Pampean region of Argentina. Plants of this species showing leaf mosaic symptoms were collected near sunflower (Helianthus annuus L.) crops infected with Sunflower chlorotic mottle virus (SuCMoV). Symptomatic plants were tested by biological, serological and molecular assays. Sunflower and Nicotiana occidentalis L. plants mechanically inoculated with extracts from symptomatic D. fullonum leaves developed typical SuCMoV symptoms. Samples were DAS‐ELISA positive when probed with a SuCMoV antiserum. RT‐PCR was used to amplify the whole coat protein gene of the infecting virus. An 807 bp fragment was amplified, cloned and sequenced. The deduced amino acid sequence (EU606023) shared 96% amino acid identity with the SuCMoV sequence (AF255677). These results indicate that D. fullonum mosaic is caused by an isolate of SuCMoV. Thus, this finding has epidemiological relevance as the weed may act as a natural virus reservoir.

Bidens Mosaic Virus is a Member of the Potato Virus Y Species

The nucleotide sequence of the genomic 3' terminal region (1,702 bases) of two Brazilian Bidens mosaic virus isolates (BiMV: BiMV-p and BiMV-b) was determined. BiMV-p and BiMV-b share 98% nucleotide sequence identity, and are most closely related to members of the potyvirus species: Sunflower chlorotic mottle virus (an isolate of Potato virus Y) and Potato virus Y. BiMV-p shares 88% capsid protein amino acid identity and 77% 3'UTR nucleotide sequence identity with SuCMoV an isolate of Sunflower chlorotic mottle virus. Phylogenetic analyses suggest the close evolutionary relationship of BiMV, SuCMoV and Potato virus Y (PVY), members of the PVY species. According to the analyses of capsid protein and 3'UTR sequences BiMV isolates must be regarded as a strain of Potato virus Y species, but their differences in host reactions and the phylogenetic distance suggest that they would be most likely better placed in a taxon between species and strains.

Sunflower Chlorotic Mottle Virus in Compatible Interactions with Sunflower: ROS Generation and Antioxidant Response

Sunflower chlorotic mottle virus (SuCMoV) is a recently described potyvirus that causes systemic infections in sunflower plants leading to chlorotic mottling and important growth reductions and yield losses. Oxidative damage is expressed after symptom development in this host-pathogen combination. The involvement of antioxidant enzyme activities in disease susceptibility was studied in two sunflower lines differing in the intensity and rate of development of SuCMoV infections: L2 is more susceptible than L33. A transient superoxide production peak was detected in leaves of both lines before symptom development. H2O2 accumulation increased before symptom expression in infected plants of L33 but in L2 such increase was registered only after symptoms became evident. In healthy plants of both lines, superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) showed similar activity levels. In inoculated plants of line L2, but not in L33, SOD and CAT activities increased significantly before the appearance of symptoms, and APX increases were detected later. A 1 mM SA treatment effectively decreased SuCMoV accumulation in plants of L2 but it did not affect it in L33. This treatment increased H2O2 accumulation and prevented the increase in antioxidant enzyme activities in infected plants of L2. It is suggested that increases in antioxidant enzyme activities interrupted the signals generated by the increase in ROS, which may have otherwise triggered defence reactions in the host and thus, resulted in a compatible interaction.

Screening Sunflower for Resistance to Sunflower chlorotic mottle virus and Mapping the Rcmo‐1 Resistance Gene

Sunflower chlorotic mottling is an emerging virus disease which has been detected in commercial hybrids and wild sunflowers in Argentina. Two hundred thirty‐two sunflower (Helianthus annuus L.) lines were screened for resistance to Sunflower chlorotic mottle virus (SuCMoV) using artificial inoculations under greenhouse conditions. Only three lines (L33, L74, and L52) showed a partial resistance, L33 being the most resistant. Virus replication was delayed (A405 in DAS‐ELISA) and morphological traits (plant height and leaf width) were less affected in this line and its various hybrid combinations than in susceptible controls. Segregation data from an artificially inoculated F2 population, under greenhouse and field conditions, indicated the presence of a single dominant resistance gene which was designated Rcmo‐1 This gene was mapped on Linkage Group 14 between markers MS0022 (5 cM) and ORS‐307 (4 cM). Linkage between the two markers and the Rcmo‐1 locus was confirmed in a field evaluation.