Beet Curly Top Iran Virus Research Articles

Eco‐friendly chitosan polymer mitigates disease severity and mediates plant resistance against Beet curly top Iran virus in tomato

AbstractA tomato‐infecting virus known as Beet curly top Iran virus (BCTIV) cause a significant disease for tomato plants and several other plant species around the world. Chitosan polysaccharide is a natural biopolymer that has been utilised as an exo‐elicitor to enhance plant defence mechanisms against a variety of plant diseases. This study investigates the efficacy of chitosan in combating BCTIV disease on tomato plants and modulating the host–virus interaction under greenhouse conditions. Twenty‐four hours before the virus inoculation, tomato plants were sprayed with a protective chitosan solution at different concentrations (0.5, 1, 1.5, and 2 mg/mL). Tomato plants were inoculated with a BCTIV infectious clone using an Agrobacterium‐inoculation method. The findings clearly demonstrated a reduction in the severity of the disease in chitosan‐treated plants as compared to Mock‐plants, with the percentage decreasing from 61.53% to 75.28% in 1.5 mg/mL treated plants and from 9.01% to 28.43% in 0.5 mg/mL treated plants. In addition, the utilisation of chitosan has the potential to deactivate the accumulation of BCTIV within the host tissues. The virus accumulation was greatly alleviated in 1, 1.5, and 2 mg/mL‐treated plants by 71.29%, 90.11%, and 93.14%, respectively, and over the mock plants. Furthermore, it was found that chitosan applied at all tested concentrations increased the relative expression and mRNA accumulation of genes related to resistance, including the pathogenesis‐related protein gene PR‐1, the HSP90 gene, and the AGO2a antiviral gene. These genes reached their maximum by 22.9‐, 12.93‐, and 4.44‐fold increases, respectively, over the untreated control. According to gas chromatography–mass spectroscop (GC‐MS) fractionation profile, chitosan increased 28 bioactive metabolic components, such as n‐hexadecanoic acid, heptanone, 1,2‐dimethylbenzene, dicarboxylic acid, and cis‐11‐octadecenoic acid methyl‐ester, to improve metabolic pathways. Results reported here revealed that foliar application of chitosan decreases the rate of the disease severity and virus accumulation in BCTIV‐infected tomato plants. This effect is associated with increased gene expression and defence‐related factors, enhancing tomato resistance to BCTIV infection. Consequently, chitosan treatments could be part of an integrated approach for reducing the severity of BCTIV disease in tomato and other host plants.

Screening of coriander (Coriandrum sativum L.) genotypes to beet curly top virus-severe and beet curly top Iran virus infection

The beet curly top virus-severe and beet curly top Iran virus are the main causal agents of curly top disease (CTD) in Iran. CTD has been described for a long time as one of the viral diseases of coriander that causes severe losses in seed production. An effective approach to managing plant viral diseases is to introduce resistant cultivars. Therefore, this study aimed to identify resistant coriander accessions for CTD. The study employed a completely randomized design with 10 replications to investigate the reaction of 10 coriander accessions, which were inoculated using agroinoculation. The inoculated plants were analyzed by measuring phenotypic, physiological and biochemical parameters, and virus accumulation. The results revealed that phenolic compounds, phenylalanine ammonia-lyase, polyphenol oxidase activity, guaiacol peroxidase, total chlorophyll content, and photosynthesis rate were elevated in resistant accessions to cope with virus inoculation. Resistant accessions exhibited normal growth and seed production similar to their mock-inoculated controls. Through principal component analysis and hierarchical cluster analysis, it was confirmed that accessions 111,230 and 230,160 were highly resistant to both viruses. These findings suggest that these accessions can be utilized in breeding programs to develop virus-resistant cultivars.

Beet Curly Top Iran Virus Rep and V2 Suppress Post-Transcriptional Gene Silencing via Distinct Modes of Action.

Beet curly top Iran virus (BCTIV) is a yield-limiting geminivirus belonging to the becurtovirus genus. The genome organization of BCTIV is unique such that the complementary strand of BCTIV resembles Mastrevirus, whereas the virion strand organization is similar to the Curtovirus genus. Geminiviruses are known to avoid the plant defense system by suppressing the RNA interference mechanisms both at the transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS) levels. Multiple geminivirus genes have been identified as viral suppressors of RNA silencing (VSR) but VSR activity remains mostly elusive in becurtoviruses. We found that BCTIV-V2 and -Rep could suppress specific Sense-PTGS mechanisms with distinct efficiencies depending on the nature of the silencing inducer and the target gene. Local silencing induced by GFP inverted repeat (IR) could not be suppressed by V2 but was partially reduced by Rep. Accordingly, we documented that Rep but not V2 could suppress systemic silencing induced by GFP-IR. In addition, we showed that the VSR activity of Rep was partly regulated by RNA-dependent RNA Polymerase 6 (RDR6), whereas the VSR activity of V2 was independent of RDR6. Domain mapping for Rep showed that an intact Rep protein was required for the suppression of PTGS. In summary, we showed that BCTIV-Rep and -V2 function as silencing suppressors with distinct modes of action.

Development of Highly Efficient Resistance to Beet Curly Top Iran Virus (Becurtovirus) in Sugar Beet (B. vulgaris) via CRISPR/Cas9 System.

Beet Curly Top Iran Virus (BCTIV, Becurtovirus) is a dominant and widespread pathogen responsible for great damage and yield reduction in sugar beet production in the Mediterranean and Middle East. CRISPR-based gene editing is a versatile tool that has been successfully used in plants to improve resistance against many viral pathogens. In this study, the efficiency of gRNA/Cas9 constructs targeting the expressed genes of BCTIV was assessed in sugar beet leaves by their transient expression. Almost all positive control sugar beets revealed systemic infection and severe disease symptoms (90%), with a great biomass reduction (68%) after BCTIV agroinoculation. On the other hand, sugar beets co-agronioculated with BCTIV and gRNA/Cas9 indicated much lower systemic infection (10-55%), disease symptoms and biomass reduction (13-45%). Viral inactivation was also verified by RCA and qPCR assays for gRNA/Cas9 treated sugar beets. PCR-RE digestion and sequencing assays confirmed the gRNA/Cas9-mediated INDEL mutations at the target sites of the BCTIV genome and represented high efficiencies (53-88%), especially for those targeting BCTIV's movement gene and its overlapping region between capsid and ssDNA regulator genes. A multiplex CRISPR approach was also tested. The most effective four gRNAs targeting all the genes of BCTIV were cloned into a Cas9-containing vector and agroinoculated into virus-infected sugar beet leaves. The results of this multiplex CRISPR system revealed almost complete viral resistance with inhibition of systemic infection and mutant escape. This is the first report of CRSIPR-mediated broad-spectrum resistance against Becurtovirus in sugar beet.

Functional Analysis of V2 Protein of Beet Curly Top Iran Virus.

Geminivirus beet curly top Iran virus (BCTIV) is one of the main causal agents of the beet curly top disease in Iran and the newly established Becurtovirus genus type species. Although the biological features of known becurtoviruses are similar to those of curtoviruses, they only share a limited sequence identity, and no information is available on the function of their viral genes. In this work, we demonstrate that BCTIV V2, as the curtoviral V2, is also a local silencing suppressor in Nicotiana benthamiana and can delay the systemic silencing spreading, although it cannot block the cell-to-cell movement of the silencing signal to adjacent cells. BCTIV V2 shows the same subcellular localization as curtoviral V2, being detected in the nucleus and perinuclear region, and its ectopic expression from a PVX-derived vector also causes the induction of necrotic lesions in N. benthamiana, such as the ones produced during the HR, both at the local and systemic levels. The results from the infection of N. benthamiana with a V2 BCTIV mutant showed that V2 is required for systemic infection, but not for viral replication, in a local infection. Considering all these results, we can conclude that BCTIV V2 is a functional homologue of curtoviral V2 and plays a crucial role in viral pathogenicity and systemic movement.

The Rep and C1 of Beet curly top Iran virus represent pathogenicity factors and induce hypersensitive response in Nicotiana benthamiana plants

Beet curly top Iran virus (BCTIV) is a member of the genus Becurtovirus (Family Geminiviridae) with a circular single-strand DNA genome. BCTIV causes leaf curling and vein swelling symptoms in plants. However, the potential pathogenicity factor/s in BCTIV is/are not known. This study presents characterization of complementary-sense transcripts of BCTIV and the viral factors in directing the pathogenicity and hypersensitive response (HR) in Nicotiana benthamiana plants. In both local and systemic infection, splicing of the complementary transcripts of BCTIV was observed. Notably, a small number (8.3%) of transcripts were spliced to produce Rep (C1:C2) transcripts after deletion of 155 nt (position 1892-2046 from BCTIV). Expression of BCTIV genes in N. benthamiana using tobacco rattle virus (TRV)-based vector showed that Rep together with C1 are the main pathogenicity factors which cause typical viral leaf curling symptoms. In addition, the V2 caused a mild leaf curling, thickening, and asymmetric leaves, while the V1, V3, and C2 had no clear effect on the plant phenotype. Transient expression of individual viral genes showed that both the C1 and Rep trigger a HR response in N. benthamiana. The higher expression of HR marker genes, harpin-induced 1 (Hin1) and hypersensitivity-related (Hsr203JI), supported the role of C1 and Rep in HR response in plants. It is concluded that Rep and C1 are the main pathogenicity factors that also trigger HR response in plants.

Beet curly top Iran virus

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Genome-based identification of beet curly top Iran virus infecting sugar beet in Turkey and investigation of its pathogenicity by agroinfection

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.

Characterization of Beet curly top Iran virus infecting eggplant and pepper in north-eastern Iran

Beet curly top Iran virus (BCTIV) (Becurtovirus, Geminiviridae) is a destructive virus in crop growing farms in Iran. In this study, BCTIV infected eggplant and chili and bell pepper plants showing typical geminivirus symptoms were collected in north-eastern Iran and investigated. PCR and sequence of the resulting amplicons showed that the samples are infected with the BCTIV. A bell pepper isolate was chosen and BCTIV full-length genome was amplified using rolling circle amplification (RCA) method. An infectious clone containing a partial dimer of the BCTIV genome was constructed and used for agroinoculation tests. Results indicated that inoculated eggplant and bell pepper seedlings with BCTIV infectious construct are efficiently infected and resulted in 60 and 47% infection. Based on the results of this study, eggplant and chili and bell peppers are the BCTIV hosts in north-eastern Iran and therefore, eggplant is reported as new natural host of BCTIV. This finding confirms broad host range of the virus.

Arbuscular mycorrhizal symbiosis enhances virus accumulation and attenuates resistance-related gene expression in tomato plants infected with Beet curly top Iran virus

Beet curly top Iran virus (BCTIV), a member of the genus Becurtovirus, is one of the causal agents for curly top disease in tomato plants. Arbuscular mycorrhizal (AM) fungi provide nutrients for host plants and are associated with their improved growth. However, the impact of AM on infection by plant viruses is not well understood. In this study, the interaction between Funneliformis mosseae and BCTIV in a susceptible tomato cultivar (Early Eurbana) was investigated. In a completely randomized design experiment, tomato seedlings were inoculated with F. mosseae, and after 4 weeks, they were inoculated with an infectious clone of BCTIV. Four treatments were included: untreated control plants (C), BCTIV-infected plants (V), mycorrhizal plants (M) and BCTIV-infected mycorrhizal plants (MV). Results of symptom evaluation based on a disease severity index showed a higher disease severity in MV plants compared to V plants. Supporting this result, a higher level of virus accumulation was observed in MV plants and this became more significant after long-term infection. The expression of three defense-related genes including HSP90, RLK and PRP1 was attenuated in MV plants compared to V plants, which may explain the enhanced symptom production and viral accumulation in these plants. A similar percentage of root colonization by F. mosseae in M and MV plants indicated that root colonization was not affected by BCTIV infection. These results show that mycorrhizal symbiosis increases the susceptibility of tomato plants to virus infection and favors BCTIV accumulation and symptom production.

Virus-mediated export of chromosomal DNA in plants

The propensity of viruses to acquire genetic material from relatives and possibly from infected hosts makes them excellent candidates as vectors for horizontal gene transfer. However, virus-mediated acquisition of host genetic material, as deduced from historical events, appears to be rare. Here, we report spontaneous and surprisingly efficient generation of hybrid virus/host DNA molecules in the form of minicircles during infection of Beta vulgaris by Beet curly top Iran virus (BCTIV), a single-stranded DNA virus. The hybrid minicircles replicate, become encapsidated into viral particles, and spread systemically throughout infected plants in parallel with the viral infection. Importantly, when co-infected with BCTIV, B. vulgaris DNA captured in minicircles replicates and is transcribed in other plant species that are sensitive to BCTIV infection. Thus, we have likely documented in real time the initial steps of a possible path of virus-mediated horizontal transfer of chromosomal DNA between plant species.

Seed Transmission of Beet Curly Top Virus and Beet Curly Top Iran Virus in a Local Cultivar of Petunia in Iran.

Beet curly top virus (BCTV) and beet curly top Iran virus (BCTIV) are known as the causal agents of curly top disease in beet and several other dicotyledonous plants in Iran. These viruses are transmitted by Circulifer species, and until now, there has been no confirmed report of their seed transmission. A percentage (38.2–78.0%) of the seedlings developed from the seeds of a petunia local cultivar under insect-free conditions showed stunting, interveinal chlorosis, leaf curling, and vein swelling symptoms, and were infected by BCTV when tested by PCR. Presence of BCTV in seed extracts of petunia local cultivar was confirmed by PCR and IC-PCR, followed by sequencing. Agroinoculation of curly top free petunia plants with a BCTV infectious clone resulted in BCTV infection of plants and their developed seeds. These results show the seed infection and transmission of BCTV in a local cultivar of petunia. Similar experiments performed with BCTIV showed that this virus is also seed transmissible in the same cultivar of petunia, although with a lower rate (8.8–18.5%). Seed transmission of curly top viruses may have significant implications in the epidemiology of these viruses.

Genome Sequences of Beet curly top Iran virus, Oat dwarf virus, Turnip curly top virus, and Wheat dwarf virus Identified in Leafhoppers.

ABSTRACTImplementation of a vector-enabled metagenomics approach resulted in the identification of various geminiviruses. We identified the genome sequences of Beet curly top Iran virus, Turnip curly top viruses, Oat dwarf viruses, the first from Iran, and Wheat dwarf virus from leafhoppers feeding on beet, parsley, pumpkin, and turnip plants.

Splicing features in the expression of the complementary-sense genes of Beet curly top Iran virus.

Beet curly top Iran virus (BCTIV) is a distinct geminivirus which has been reported from sugar-beet-growing farms in Iran. In this study, the role of the splicing in expression of complementary-sense genes of BCTIV was studied. Total RNA was extracted from BCTIV-infected tissue, and the predicted intron position of complementary-sense mRNA transcripts was amplified by RT-PCR followed by cloning of the amplicons. Sequence confirmed that both spliced and unspliced mRNAs are synthesized by the same transcription unit. Sequence comparison showed that a 155-nt segment (intron) corresponding to nucleotides 1890-2044 of the viral genome has been removed from the latter transcript and therefore fusion of the C1:C2 genes resulted creation of a continuous reading frame for potential production of intact replication initiator protein (Rep). BCTIV intron comprises of most consensus splicing signals required for splicing in eukaryotes and several plant viruses including mastre- and capulaviruses.

In silico analysis of microRNA binding to the genome of Beet curly top Iran virus in tomato

Beet curly top Iran virus (BCTIV) is a new member of geminiviruses causing leaf curl disease in a wide range of crops. These DNA viruses can both induce and be themselves targeted by gene silencing machinery. In animals, miRNAs were found to limit viral establishment by direct interference with viral replication or by targeting viral transcripts. However, such mechanisms have not been yet reported for plant viruses. In this work, we investigated the possible binding of tomato encoded miRNA/miRNA* sequences to the viral genome and/or transcripts of BCTIV. In silico analysis showed that several miRNA/miRNA* species including mir319, mir1919 and mir159 can potentially target both the genome and transcribed ORFs of the BCTIV. In addition, the identified miRNAs* also were predicted to bind host genes from various metabolic pathways. This may suggest a novel role for plant miRNA/miRNA* in host-virus interactions and defence response.

Response of tomato cultivars to agroinfection with Beet curly top Iran virus

Beet curly top Iran virus (BCTIV) is a member of the genus Becurtovirus (family Geminiviridae) which constrain host crop production in various geographical regions in Iran. This virus infects several crops such as sugar beet Beta vulgaris and tomato Solanum lycopersicum. BCTIV infected tomato shows leaf curling, leaf distortion and stunting. In this study, we investigated the response of various tomato cultivars to BCTIV infection using an infectious clone of BCTIV under glasshouse condition at the University of Zanjan in 2013-2014. Based on a completely randomized design experiment twelve tomato cultivars were tested for their reaction to BCTIV infection. The replication of virus and symptom production was recorded and analyzed. Based on the obtained coefficient of infection and disease severity index, none of the tested cultivars was found resistance to the virus infection. However, one of the tested cultivars, Super Chief, showed no leaf curling symptom and the virus replicated at a significantly lower level in this cultivar as compared to a susceptible cultivar, Alindi 811, using quantitative PCR. Tomato cultivars including Grosse Lisse and Early Eurbana were grouped as susceptible while the other cultivars including Super Star were moderately susceptible to the virus infection. Therefore, growing this cultivar shows promise for an increase of yields from tomato plants prone to BCTIV infection after complementary field experiments. The screening of more cultivars or wild-type tomatoes for the identification of gene/s providing resistance to this viral disease is suggested.

Molecular characterization and construction of an infectious clone of a pepper isolate of Beet curly top Iran virus.

Geminiviruses cause curly top disease, in dicotyledonous plants which constrains host crop production. Beet curly top Iran virus (BCTIV) is a widespread Becurtovirus (family Geminiviridae) in numerous areas within Iran. In this study, we isolated and analyzed a full-length genomic DNA of a new variant of BCTIV from pepper crops in the Kaftark region, east of Shiraz (proposed acronym: BCTIV-Kaf [IR: Kaf:2016:Pepper]). Infected pepper plants showed shortening of internodes, severe interveinal chlorosis, upward leaf rolling and leaf curling. Sequence and phylogenetic analysis showed this BCTIV variant grouped with sugar beet isolates of BCTIV and has the highest similarity to a sugar beet BCTIV isolate from Negar town in Kerman province, Iran. It was more distantly related to a bean isolate of BCTIV from northeast region of Iran. A tandem repeat partial dimmer of BCTIV was constructed and found to be infectious in pepper, tomato and Nicotiana benthamiana plants. Results of this study indicated that BCTIV-Kaf is a new variant of BCTIV infecting pepper plants in Shiraz and that geographic location rather than the type of host plant has more effect on genetic diversity of BCTIV in Iran.

Evaluation of sugar beet lines for resistance to beet curly top viruses

Curly top disease caused by beet curly top viruses (BCTVs) has been a problem in sugar beet fields for a long time. In recent years, climate change has increased the spread of virus vectors and disease damage in infected regions. To find a natural resistance source to curly top viruses in Iran (Beet curly top virus-C, BCTV-C and Beet curly top Iran virus, BCTIV), the reaction of 50 sugar beet lines to the above-mentioned viruses was evaluated in a completely randomized design experiment in 2014. Sugar beet lines were agroinoculated at the 4–6 leaf stage by virus infectious clones under greenhouse condition and then the symptom severity and virus presence was monitored by enzyme-linked immunosorbent assay and polymerase chain reaction methods 8 weeks after inoculation. Five out of 50 lines were found to be resistant to both viruses. Given that both viruses were detected in most sugar beet cultivation areas in Iran, these lines could be suggested for cultivation in Iran. In addition, some lines displayed completely different reactions to the viruses indicating different effects of the viruses on sugar beet lines.

Establishment of three new genera in the family Geminiviridae: Becurtovirus, Eragrovirus and Turncurtovirus

The family Geminiviridae includes plant-infecting circular single-stranded DNA viruses that have geminate particle morphology. Members of this family infect both monocotyledonous and dicotyledonous plants and have a nearly global distribution. With the advent of new molecular tools and low-cost sequencing, there has been a significant increase in the discovery of new geminiviruses in various cultivated and non-cultivated plants. In this communication, we highlight the establishment of three new genera (Becurtovirus, Eragrovirus and Turncurtovirus) to accommodate various recently discovered geminiviruses that are highly divergent and, in some cases, have unique genome architectures. The genus Becurtovirus has two viral species, Beet curly top Iran virus (28 isolates; leafhopper vector Circulifer haematoceps) and Spinach curly top Arizona virus (1 isolate; unknown vector), whereas the genera Eragrovirus and Turncurtovirus each have a single assigned species: Eragrostis curvula streak virus (6 isolates; unknown vector) and Turnip curly top virus (20 isolates; leafhopper vector Circulifer haematoceps), respectively. Based on analysis of all of the genome sequences available in public databases for each of the three new genera, we provide guidelines and protocols for species and strain classification within these three new genera.

Intergeneric recombination between a new, spinach-infecting curtovirus and a new geminivirus belonging to the genus Becurtovirus: first New World exemplar

A novel curtovirus, spinach severe curly top virus (SSCTV), was associated with symptomatic spinach plants collected from a commercial field in south-central Arizona during 2009. In addition, a second viral molecule of about 2.9 kb from the same spinach plants was amplified, cloned and sequenced. The latter isolate, herein named spinach curly top Arizona virus (SCTAV), was found to share 77 % pairwise sequence identity with beet curly top Iran virus (BCTIV), a leafhopper-transmitted geminivirus that has been assigned to the new genus Becurtovirus. The SCTAV genome encodes three viral-sense genes, V1, V2, and V3, and two complementary-sense genes, C1 and C2. There was no evidence for the presence of either a C3 or C4 ORF in the genome sequence. The genome organization of SCTAV is not like that of New World curtoviruses but instead is similar to that of BCTIV, which, to date, is only known to be present in Iran. Consistent with this observation, SCTAV and BCTIV both contain the unusual nonanucleotide TAAGATT/CC and a replication-associated protein, Rep (or C1), that is more closely related to the mastrevirus Rep than to those of curtoviruses reported to date. Both SSCTV and SCTAV were found to have a recombinant genome containing sequences (AY548948) derived from ancestral SCTV sequences in the virion-sense portions of the genome. Agroinoculation of Nicotiana benthamiana (Domin) plants with the cloned genome of SCTAV resulted in infection of 95 % of the plants and the development of severe curling symptoms, whereas only 20 % of the SSCTV-inoculated plants were infected, developing only mild curling symptoms. When plants were co-inoculated with both viruses, the frequency of infection remained higher for SCTAV than for SSCTV (80 % vs. 20 %), indicating no evidence of synergistic effects between the two viruses with respect to efficiency of infection.