The Quest to Identify a New Virus Disease of Sunflower 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.

Direct Metatranscriptomic Survey of the Sunflower Microbiome and Virome.

Sunflowers (Helianthus annuus L.) are susceptible to multiple diseases in field production. In this study, we collected diseased sunflower leaves in fields located in South Dakota, USA, for virome investigation. The leaves showed visible symptoms on the foliage, indicating phomopsis and rust infections. To identify the viruses potentially associated with the disease diagnosed, symptomatic leaves were obtained from diseased plants. Total RNA was extracted corresponding to each disease diagnosed to generate libraries for paired-end high throughput sequencing. Short sequencing reads were assembled de novo and the contigs with similarities to viruses were identified by aligning against a custom protein database. We report the discovery of two novel mitoviruses, four novel partitiviruses, one novel victorivirus, and nine novel totiviruses based on similarities to RNA-dependent RNA polymerases and capsid proteins. Contigs similar to bean yellow mosaic virus and Sclerotinia sclerotiorum hypovirulence-associated DNA virus were also detected. To the best of our knowledge, this is the first report of direct metatranscriptomics discovery of viruses associated with fungal infections of sunflowers bypassing culturing. These newly discovered viruses represent a natural genetic resource from which we can further develop potential biopesticide to control sunflower diseases.

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.

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.

Occurrence and genetic diversity of the Plasmopara halstedii virus in sunflower downy mildew populations of the world.

Plasmopara halstedii virus (PhV) is a ss(+)RNA virus that exclusively occurs in the sunflower downy mildew pathogen Plasmopara halstedii, a biotrophic oomycete of severe economic impact. The virus origin and its genomic variability are unknown. A PCR-based screening of 128 samples of P. halstedii from five continents and up to 40 y old was conducted. PhV RNA was found in over 90 % of the isolates with no correlation to geographic origin or pathotype of its host. Sequence analyses of the two open reading frames (ORFs) revealed only 18 single nucleotide polymorphisms (SNPs) in 3873 nucleotides. The SNPs had no recognizable effect on the two encoded virus proteins. In 398 nucleotides of the untranslated regions (UTRs) of the RNA 2 strand eight additional SNPs and one short deletion was found. Modelling experiments revealed no effects of these variations on the secondary structure of the RNA. The results showed the presence of PhV in P. halstedii isolates of global origin and the existence of the virus since more than 40 y. The virus genome revealed a surprisingly low variation in both coding and noncoding parts. No sequence differences were correlated with host pathotype or geographic populations of the oomycete.

Plasmopara halstedii virus causes hypovirulence in Plasmopara halstedii, the downy mildew pathogen of the sunflower.

Plasmopara halstedii virus (PhV) is an isometric virus recently found in the oomycete Plasmopara halstedii. The fully sequenced virus genome consists of two ss(+)RNA strands encoding for the virus polymerase and the coat protein, respectively. Most of previously screened field isolates of P. halstedii were found to harbor PhV, but effects of PhV on the pathogenicity and aggressiveness of the oomycete have not been investigated yet. To assess the influence of PhV on the infectivity of P. halstedii, virus-free isolates of the oomycete were searched for, cultivated on sunflower and used for single zoospore infection. Four genetically homogenous strains belonging to three different pathotypes (710, 730, 750) were established. Subcultures of each strain were successfully infected with PhV. This afforded pairs of isogenic strains with and without virus and allowed assessment of the pathogenicity (susceptibility to specific sunflower genotypes) and aggressiveness (intensity of infection, time scale and density of sporulation) in cultivation of sunflower. While no significant difference was found in the pathogenicity of P. halstedii strains with and without virus towards sunflower seedlings of different resistance (pathotype differentials), the aggressiveness of the oomycete was diminished by PhV. Compared to the virus-free strains, the time required for the first sporulation (latent period) increased by about 1 day post inoculation. Progression of the pathogen from the hypocotyl into the epicotyl of sunflower (systemic infection) was reduced by about one third in the presence of virus. In the virus containing strains, the average density of sporangia produced per cm² cotyledon reached only 75% of the virus-free controls. In summary, the presence of PhV leads to hypovirulence effects by weakening the aggressiveness of P. halstedii.

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.

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.

Development of a probe-based blotting technique for the detection of tobacco streak virus.

Digoxigenin (DIG)-labeled DNA probe was developed for a sensitive and rapid detection of the tobacco streak virus (TSV) isolates in India by dot-blot and tissue print hybridization techniques. DIG-labeled DNA probe complementary to the coat protein (CP) region of TSV sunflower isolate was designed and used to detect the TSV presence at field levels. Dot-blot hybridization was used to check a large number of TSV isolates with a single probe. In addition, a sensitivity of the technique was examined with the different sample extraction methods. Another technique, the tissue blot hybridization offered a simple, reliable procedure and did not require a sample processing. Thus, both non-radioactively labeled probe techniques could facilitate the sample screening during TSV outbreaks and offer an advantage in quarantine services.

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.

Full-length sequence analysis of a distinct isolate of Bidens mottle virus infecting sunflower in Taiwan.

The full-length genome of a potyvirus, previously known as sunflower chlorotic spot virus isolate SF-1 (SCSV-SF-1) which causes novel symptoms on sunflowers (Helianthus annuus), was sequenced and analyzed. The genome of SCSV-SF-1 is 9,741 nucleotides long, encoding a polyprotein of 3,071 amino acids containing the consensus motifs of potyviruses. Sequence comparison revealed that the 3'-terminus of SCSV-SF-1 shared over 96% similarities with isolates of Bidens mottle virus (BiMoV). However, SCSV-SF-1 has a very narrow host range, excluding the diagnostic host species for BiMoV, Bidens pilosa and Zinnia elegans. Therefore, SCSV-SF-1 is a distinct isolate of BiMoV. This is the first report of the full-length nucleotide sequence of BiMoV infecting sunflower in Taiwan.

Serological and coat protein sequence studies suggest that necrosis disease on sunflower in India is caused by a strain of Tobacco streak ilarvirus.

Serological and coat protein sequence studies were conducted to identify an ilarvirus associated with necrosis disease on sunflower in India. In electroblot immunoassay, sunflower ilarvirus reacted strongly only with antiserum to Tobacco streak virus (TSV). The coat protein gene of sunflower ilarvirus was cloned and sequenced. The sequence analyses also showed that the CP gene was most closely related to TSV, the member of subgroup I of Ilarvirus. The sunflower ilarvirus CP shared 90% amino acid sequence identity with TSV. On the basis of serological relatedness and sequence identity, it is proposed that the sunflower ilarvirus from India should be considered a strain of TSV belonging to subgroup I and designated as TSV-SF. This is the first report of the molecular characterization of TSV on sunflower from the Indian subcontinent.

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.

[Isolation and purification of virus damaging sunflower]. / Vydilennia ta ochystka virusu, shcho soniashnyk.

A procedure has been developed for purifying intact virus's isolate particles evoking yellow spot mosaic disease in sunflower. Purification of pathogen in 0.1 M sodium phosphate buffer, pH 8.0 containing 0.05 M Na3SO3 and 0.2% 2-mercaptoethanol is used. After first clarification extract was exposed to two cycles of high-speed centrifugation and fractionated in linear 10-40% (wt vol-1) sucrose density gradient. Virus was recovered from appropriate fractions after dialysis against 0.01 M Na2SO3.