Beet Curly Top Iran Virus Research Articles

Diversity of Beet curly top Iran virus isolated from different hosts in Iran

Beet curly top Iran virus (BCTIV) is a major pathogen of sugar beet in Iran. In order to study diversity of BCTIV, we sampled 68 plants in Iran during the summer of 2010 with curly top disease symptoms on beans (Phaseolus vulgaris), cowpeas (Vigna unguiculata), tomatoes (Solanum lycopersicum L.), sea beets (Beta vulgaris subsp. maritima), and sugar beets (Beta vulgaris). Plant samples showing leaf curling, yellowing, and/or swelling of veins on the lower leaf surfaces were collected from various fields in Khorasan Razavi, Northern Khorasan (north-eastern Iran), East Azarbayejan, West Azarbayejan (north-western Iran), and Fars (southern Iran) provinces. Using rolling circle amplification coupled with restriction digests, cloning, and Sanger sequencing, we determined the genomes of nine new BCTIV isolates from bean, cowpea, tomato, sea beet, and sugar beet in Iran. Our analysis reveals ~11 % diversity amongst BCTIV isolates and we detect evidence of recombination within these genomes.

The unconventional geminivirus Beet curly top Iran virus: satisfying Koch's postulates and determining vector and host range

AbstractBeet curly top Iran virus (BCTIRV) is a geminivirus with unusual genomic organisation, recently reported in Iran, infecting sugarbeet and a few other plant species. Although three BCTIRV sequences have been reported, demonstration that BCTIRV DNA is the causal agent of the disease was missing. A full‐length genomic DNA was obtained from symptomatic leaves of sugarbeet collected in the Sivand area of Iran, and its nucleotide sequence was determined (BCTIRV‐Siv, 2845 nt). To satisfy Koch's postulates, an infectivity assay was developed by inserting a 1.4‐mer of BCTIRV‐Siv DNA in Agrobacterium tumefaciens and using it in agroinoculation experiments. The cloned viral DNA was capable of infecting sugarbeets, reproducing the leaf curling and vein enations observed in the field. These results demonstrate that the single DNA component of BCTIRV is sufficient for infectivity. Host range studies indicated that some economically important crops can be affected, such as spinach, tomato and sweet pepper, as well as important laboratory plants including Nicotiana benthamiana, Arabidopsis thaliana and Jimson weed. Circulifer haematoceps, the dominant leafhopper species present in sugarbeet fields in Iran, was successfully used to transmit the disease. The availability of an infectious clone will facilitate extended host range studies, to determine the potential risks to other crops, as well as genetic studies on this unusual member of the family Geminiviridae.

Fulfilling Koch’s postulates for beet curly top Iran virus and proposal for consideration of new genus in the family Geminiviridae

Beet curly top Iran virus (BCTIV) is a divergent geminivirus with biological properties similar to those of curtoviruses; however, the virus is distinct from curtoviruses phylogenetically and in its genome organisation. The replication-associated protein is phylogenetically more closely related to those of mastreviruses than to those of curtoviruses whereas the capsid protein shares high amino acid sequence identity (77-83%) with those of curtoviruses. The 17 BCTIV genomes from Iran share ~77% pairwise nucleotide sequence identity with spinach curly top Arizona virus (SCTAV) from Arizona, USA, which was characterised recently. To demonstrate the infectivity of the monopartite BCTIV genome and to fulfil Koch's postulates, an infectious clone was constructed using a dimer of the full-length genome of an isolate from this study - BCTIV-[IR:Neg:B33P:Sug:08]. Agroinoculation with the cloned DNA resulted in the efficient infection of 74% of sugar beet plants, which resulted in curly top symptoms. The curly top infection of agroinoculated plants was successfully transmitted to 80% of healthy sugar beet plants by the natural BCTIV vector, Circulifer haematoceps. Since BCTIV and SCTAV share <62% pairwise nucleotide sequence identity with all other geminiviruses and have unique genome architectures and properties, and since this is coupled with phylogenetic support at the full-genome level and that of it proteins, we propose that they should be re-classified as members of a new genus, "Becurtovirus", in the family Geminiviridae.

First Report of a New Curtovirus Species, Spinach severe curly top virus, in Commercial Spinach Plants (Spinacia oleracea) from South-Central Arizona.

During April 2009, a commercial spinach field (1 km2 [250 acres]) in south-central Arizona developed geminivirus-like disease symptoms (4). Approximately 40 to 50% of the spinach plants exhibited extreme leaf distortion, foliar interveinal chlorosis, shortened internodes, and ~80% yield reduction. The beet leafhopper, Circulifer tenellus, the only known insect vector of curtoviruses in the United States, was observed on spinach plants. Total DNA was isolated (1) from three plant samples exhibiting the same symptom phenotype and used to PCR-amplify a 446-bp fragment of a suspected curtovirus, using primers F 5'-CTACCATCAGTAATGATGGG-3'and R 5' CATATTTGCCACCTCCAGTGTC-3' designed around the coat protein gene (Cp) for several known curtoviruses. DNA sequencing and BLAST analysis of the cloned fragments (n = 3 with 100% identity) revealed BLAST matches at 81 to 83% with the Cp for three isolates of Beet curly top Iran virus (BCTIV) (EU273816-18). To amplify the full-length curtovirus genome, total DNA from one of the three positive samples was used as the template in rolling circle amplification (RCA) employing the non-sequence specific TempliPhi 100 Amplification System (GE Healthcare) that amplifies circular DNA templates. The RCA products were linearized with PstI, yielding a ~3-Kbp fragment that was cloned into pGEM3zf+ (Promega, Madison, WI). To obtain the complete sequence, one plasmid (09-10-8) containing a full-length insert was selected and prepared for sequencing with the Template Generation System II Kit (Finnzymes, Espoo, Finland). The resultant 28 sequences were assembled into a contig using SeqMan software (DNASTAR, Madison, WI). Also, RCA clones (09.10-2, -3, and -4) from the same sample were subjected to DNA sequencing with universal M13F and M13R primers followed by primer walking (>300 bp overlap). The four 3,066-bp genomes shared 99 to 100% nt identity. An alignment (ClustalV; MegAlign, DNASTAR) with sequences of all curtovirus species available in GenBank indicated that the Arizona spinach isolates shared the highest nt sequence identity (59%) with Horseradish curly top virus (HrCTV). The next closest relatives were Beet mild curly top virus, Beet severe curly top virus, and Spinach curly top virus, at 50%. The genome consists of six open reading frames and lacks the AC3 gene, an arrangement most similar to HrCTV (3). The ICTV approved working cut-off for Curtovirus species demarcation at <89% nt identity (2) supports recognition of this isolate from spinach (GU734126) as a new, previously undescribed curtovirus species, for which we propose the name Spinach severe curly top virus (SSCTV-[Arizona:2009]). The curtovirus-like symptoms, presence of the curtovirus leafhopper vector, and isolation of a curtovirus-like genome from symptomatic spinach plants are highly suggestive of curtovirus etiology. To our knowledge, this is the first report of SSCTV worldwide and its association with diseased spinach in Arizona. Although a different curtovirus species was reported from the same infected spinach field (4), this study provides evidence that at least two curtoviruses were present in this spinach field in Arizona.

A highly divergent South African geminivirus species illuminates the ancient evolutionary history of this family.

BackgroundWe have characterised a new highly divergent geminivirus species, Eragrostis curvula streak virus (ECSV), found infecting a hardy perennial South African wild grass. ECSV represents a new genus-level geminivirus lineage, and has a mixture of features normally associated with other specific geminivirus genera.ResultsWhereas the ECSV genome is predicted to express a replication associated protein (Rep) from an unspliced complementary strand transcript that is most similar to those of begomoviruses, curtoviruses and topocuviruses, its Rep also contains what is apparently a canonical retinoblastoma related protein interaction motif such as that found in mastreviruses. Similarly, while ECSV has the same unusual TAAGATTCC virion strand replication origin nonanucleotide found in another recently described divergent geminivirus, Beet curly top Iran virus (BCTIV), the rest of the transcription and replication origin is structurally more similar to those found in begomoviruses and curtoviruses than it is to those found in BCTIV and mastreviruses. ECSV also has what might be a homologue of the begomovirus transcription activator protein gene found in begomoviruses, a mastrevirus-like coat protein gene and two intergenic regions.ConclusionAlthough it superficially resembles a chimaera of geminiviruses from different genera, the ECSV genome is not obviously recombinant, implying that the features it shares with other geminiviruses are those that were probably present within the last common ancestor of these viruses. In addition to inferring how the ancestral geminivirus genome may have looked, we use the discovery of ECSV to refine various hypotheses regarding the recombinant origins of the major geminivirus lineages.

Genome characterization and genetic diversity of beet curly top Iran virus: a geminivirus with a novel nonanucleotide

Beet curly top Iran virus (BCTIV) was previously reported as a distinct curtovirus in Iran. Complete nucleotide sequences of three BCTIV isolates, one each from central, southern, and south eastern Iran were determined to be 2844, 2844, and 2845 nt long, respectively. BCTIV shared highest nucleotide sequence identity (52.3%) with Spinach curly top virus (SpCTV) and lowest identity (46.6%) with Horseradish curly top virus (HrCTV). The BCTIV genome comprises three virion-sense (V1, V2, and V3) and two complementary-sense (C1 and C2) ORFs. ORFs C3 and C4 were not found in BCTIV genome. Based on a comparison of nucleotide sequence identity of individual genes, the three virion-sense ORFs were 72.7-79.9% related to the corresponding ORFs of curtoviruses, whereas no significant relationship was found between the C1 and C2 ORFs of BCTIV and curtoviruses. These two ORFs, however, were only distantly related with those of mastreviruses. Similar to the latter viruses, the BCTIV genome comprises two intergenic regions. The BCTIV large intergenic region included a sequence capable of forming a stem loop structure and a novel nonanucleotide (TAAGATT/CC) with a unique nick site. Phylogenetic analysis using deduced amino acid sequence of individual ORFs revealed that the V2 and V3 ORFs are monophyletic and the V1 ORF is classified with the related ORF of curtoviruses. Whereas the two complementary-sense ORFs are grouped with those of mastreviruses. Computer-based prediction suggested that BCTIV has a chimeric genome which may have arisen by a recombination event involving curto- and mastrevirus ancestors. Percent nucleotide sequence identities of the coat protein gene of ten isolates of BCTIV, collected from a wide range of geographical regions in Iran, varied from 87.1 to 99.9, with the isolates being distributed between two subgroups. Based on biological and molecular properties, BCTIV is proposed as a new member of the genus Curtovirus.

P2G198 - Modulation of LD 50 and acute neurobehavioural toxicity of pyrethroid pesticide, cypermethrin, by nicotine

Beet curly top disease (BCTD) is a yield-limiting viral infection of sugar beet (Beta vulgaris) throughout the arid and semi-arid regions of the world. Two virus species, belonging to two different genera of the family Geminiviridae (Curtovirus and Becurtovirus) had been described as the disease’s causative agents on sugar beet. Despite the detection of the BCTD in some sugar beet fields of Turkey sixty years ago, the genome based characterization of BCTD-associated viruses have not been studied previously. In this study, 628 sugar beet plants exhibiting BCTD symptoms were collected from fourteen cities in central Anatolia, the major sugar beet production areas in Turkey. PCR assays of these samples using the respective Curtovirus and Becurtovirus genus-specific primers indicated that the Turkish sugar beet samples' viral sequences belong only to the genus Becurtovirus. The results of sequencing and phylogenetic analysis of the partial genome of the virus obtained from fourteen cities confirmed that BCTD-associated virus in Turkish sugar beet fields is beet curly top Iran virus (BCTIV-Becurtovirus) species. The whole genome of the collected viruses from fourteen cities were amplified by the rolling circle amplification (RCA) and the five most phylogenetically diverse viruses obtained from Afyon, Ankara, Adapazarı, Yozgat and Aksaray were sequenced. The results of whole genome sequence analysis indicated >98 % sequence identities with that of a BCTIV variants reported from Urmia province (bordering Turkey) of Iran. A virus genome from Yozgat city had a genomic sequence identity of >97 % with those of BCTIV isolated from cowpea, tomato, pepper and sugar beet in the northern part of Iran. These results suggested that the spread of BCTIV through the region could create a significant threat to the production of sugar beet as well as other agricultural crops. A tandem dimer of a BCTIV-Turkish variant isolated from Ankara city was cloned into Agrobacterium plasmid to be used for agro-infection studies. Agroinoculation of this construct on sugar beet leaves generated severe BCTD symptoms (84 %) which were also confirmed by RCA and qPCR analysis. These results constituted the first genome based characterization of BCTIV Turkish variants and the first report of BCTIV spreading out of Iran.