Vector transmission of parsley yellow leaf curl virus by the leafhopper Austroagallia sinuata.

Parsley yellow leaf curl virus (PYLCV) is a new member of the family Geminiviridae that has not yet been assigned to an established genus due to limited information about its biological properties. In this study, the ability of Austroagallia leafhoppers, which are commonly found on vegetable farms in Kerman province (Iran), to transmit this virus was studied. After a two-day acquisition access period, Austroagallia sp. successfully transmitted the virus from PYLCV-infected parsley to healthy seedlings. On the basis of male genitalia morphology, the species of leafhopper was identified as A. sinuata. This is the first report of a transmission of plant virus by a member of the genus Austroagallia.

Metatranscriptome analysis of symptomatic bitter apple plants revealed mixed viral infections with a putative novel polerovirus.

BACKGROUND: Next-generation Sequencing (NGS) combined with bioinformatic analyses constitutes a powerful approach for identifying and characterizing previously unknown viral genomes. In this study, leaf samples from bitter apple plants (Citrullus colocynthis (L.) Schrad) exhibiting symptoms such as dwarfing, leaf crinkling, and chlorosis were collected from the southern part of Kerman province, Iran. RESULTS: Putative infecting viruses were identified through de novo assembly of sequencing reads using various tools, followed by BLAST analysis. Complete genomes for Squash vein yellowing virus (SqVYV), Citrus-associated rhabdovirus (CiaRV), and a novel polerovirus-related strain termed Bitter apple aphid-borne yellows virus (BaABYV) were assembled and characterized. Additionally, a partial genome for Watermelon mosaic virus (WMV) was assembled. The genomic organization of the BaABYV was determined to be 5'-ORF0-ORF1-ORF1,2-ORF3a-ORF3-ORF3,5-ORF4-3'. Amino acid sequence identities for inferred proteins (P0 and P1, P1,2) with known poleroviruses were found to be the 90% species delineation limit, implying that BaABYV should be considered a new member of the genus Polerovirus. Recombination events were observed in the BaABYV and WMV strains; such events were not found in the CiaRV strain. CONCLUSIONS: Molecular evidence from this study suggests that C. colocynthis is a reservoir host of several plant viruses. Among them, BaABYV is proposed as a new member of the genus Polerovirus. Furthermore, the CiaRV strain has been reported for the first time from Iran.

Novel nanovirus and associated alphasatellites identified in milk vetch plants with chlorotic dwarf disease in Iran.

Members of the family Nanoviridae are multi-component single-stranded DNA viruses that infect a variety of plant species. Using a combination of conventional PCR and high throughput sequencing-based approach, we identified a novel nanovirus infecting two symptomatic milk vetch plants (Astragalus myriacanthus Boiss.; family Fabaceae) showing marginal leaf chlorosis, little leaves and dwarfing in Iran. All eight segments (DNA-C, DNA-M, DNA-N, DNA-R, DNA-S, DNA-U1, DNA-U2 and DNAU4) were recovered and Sanger sequenced. The genome of this new nanovirus, hereby referred to as milk vetch chlorotic dwarf virus (MVCDV), shares 62.2-74.7 % nucleotide pairwise identity with the genomes of other nanoviruses. DNA-C, DNA-M, DNA-N, DNA-S components are most closely related to those of black medic leaf roll virus (BMLRV), sharing between 67.8-81.2 % identity. We also identified three nanoalphasatellites (family Alphasatellitidae) associated with the nanovirus which belong to species Faba bean necrotic yellows alphasatellite 1 (genus Subclovsatellite), Faba bean necrotic yellows alphasatellite 2 (genus Fabenesatellite) and Sophora yellow stunt alphasatellite 5 (genus Clostunsatellite). Given the significant diversity of Astragalus spp. in Iran, it is likely that there could be more nanoviruses circulating in these plants and that these may play a role in the spread of these nanovirus to cultivated fabaceous hosts.

Identification of the wild and cultivated hosts of wheat dwarf virus and oat dwarf virus in Iran.

In the last decade two mastreviruses, Wheat dwarf virus (WDV) and Oat dwarf virus (ODV) have been reported from cereal farms in Iran. In a survey, wild and cultivated hosts of these mastreviruses were studied during 2015 to 2017. Symptomatic small grain cereal samples and weed species were collected and assayed for WDV and/or ODV infection by PCR. While WDV which was detected in 139/284 (49%) of total symptomatic samples, low incidence (2%) was recorded for ODV which was detected only in slender wild oat (Avena barbata Pott ex Link) and red brome (Bromus rubens L.). In agroinfection studies, the clone of ODV infected common oat (A. sativa) and slender wild oat (A. barbata) with the low efficiency and did not infect wheat or barley. ODV was transmitted by the leafhopper Psammotettix alienus, from agroinfected common oat to healthy seedlings. The results show that, in contrast to WDV, ODV has a low incidence and a narrow host range in gramineous plants.

The Westward Journey of Alfalfa Leaf Curl Virus.

Alfalfa leaf curl virus (ALCV), which causes severe disease symptoms in alfalfa (Medicago sativa L.) and is transmitted by the widespread aphid species, Aphis craccivora Koch, has been found throughout the Mediterranean basin as well as in Iran and Argentina. Here we reconstruct the evolutionary history of ALCV and attempt to determine whether the recent discovery and widespread detection of ALCV is attributable either to past diagnostic biases or to the emergence and global spread of the virus over the past few years. One hundred and twenty ALCV complete genome sequences recovered from ten countries were analyzed and four ALCV genotypes (ALCV-A, ALCV-B, ALCV-C, and ALCV-D) were clearly distinguished. We further confirm that ALCV isolates are highly recombinogenic and that recombination has been a major determinant in the origins of the various genotypes. Collectively, the sequence data support the hypothesis that, of all the analyzed locations, ALCV likely emerged and diversified in the Middle East before spreading to the western Mediterranean basin and Argentina.

Identification of a new turncurtovirus in the leafhopper Circulifer haematoceps and the host plant species Sesamum indicum.

Turncurtoviruses (family: Geminiviridae; genus: Turncurtovirus) appear to have a high degree of genetic variation in Iran. Leafhoppers of the species Circulifer haematoceps (Mulsant and Rey, 1855) (family: Cicadellidae) were collected in 2014 from three geographical regions in south-eastern Iran (Orzoeyeh, Jiroft and Sirjan; Kerman province) and screened for the presence of turncurtoviruses using a combination of PCR and rolling circle amplification (RCA) methods. Eleven genomes of turncurtovirus were recovered and sequenced. Leafhoppers were sampled off sesame (S. indicum L.) and turnip (Brassica rapa sub sp. rapa). Thus, we identified three symptomatic sesame plants (yellowing, boat-shaped leaf curling, vein swelling on the lower leaf surfaces) from sesame farms in Jiroft. In these samples, we identified the same turncurtovirus as in the leafhoppers and have named it sesame curly top virus (SeCTV). Collectively, these SeCTV share > 98% genome-wide pairwise identity and ~ 87.3% to a recently identified turncurtovirus (sesame yellow mosaic virus; SeYMV) from sesame in Pakistan (GenBank accession MF344550). The SeCTV and SeYMV sequences share < 70% genome-wide pairwise identity with isolates of Turnip curly top virus and Turnip leaf roll virus, the two species in the genus Turncurtovirus. Based on the pairwise identities and phylogenetic analysis, SeCTV (n = 12) and SeYMV (n = 1) represent two strains of a new species in the genus Turncurtovirus.

Streamlined generation of plant virus infectious clones using the pLX mini binary vectors.

Recent metagenomic surveys have provided unprecedented amounts of data that have revolutionized our understanding of virus evolution and diversity. Infectious clones are powerful tools to aid the biological characterization of viruses. We recently described the pLX vectors, a set of mini binary T-DNA vectors (∼3 kb) that includes strong bacterial terminators and a minimal replicon from the broad-host-range plasmid pBBR1, which replicate autonomously in both Escherichia coli and Agrobacterium. In this study, a workflow that encompassed pLX binary vectors, overlap-based assembly strategies, and sequencing-by-synthesis verification steps is described and applied for the streamlined generation of infectious clones suitable for Agrobacterium-mediated delivery. The pLX-based vectors herein assembled include the first infectious clone of Wasabi mottle virus, a crucifer-infecting tobamovirus, as well as binary vectors of positive-single-stranded RNA and single- and double-stranded DNA viruses from the Potyviridae, Geminiviridae and Caulimoviridae families, respectively. Finally, the clones generated were used to agro-inoculate the model plant Arabidopsis thaliana and infections were confirmed by a multiplex RT-PCR assay. This workflow facilitated the rapid generation of infectious clones which, together with agro-infection scalability, would allow the pursuit of systematic insights into virus biology and physiology of plant infections and the design of novel biotechnological applications.

Identification of a Nanovirus-Alphasatellite Complex in Sophora alopecuroides.

Viruses in the genus Nanovirus of the family Nanoviridae generally have eight individually encapsidated circular genome components and have been predominantly found infecting Fabaceae plants in Europe, Australia, Africa and Asia. For over a decade Sophora alopecuroides L. (Fabaceae) plants have been observed across Iran displaying dwarfing, yellowing, stunted leaves and yellow vein banding. Using a high-throughput sequencing approach, sequences were identified within one such plant that had similarities to nanovirus genome components. From this plant, the nanovirus-like molecules DNA-R (n=4), DNA-C (n=2), DNA-S (n=1), DNA-M (n=1), DNA-N (n=1), DNA-U1 (n=1), DNA-U2 (n=1) and DNA-U4 (n=1) were amplified, cloned and sequenced. Other than for the DNA-R, these components share less than 71% identity with those of other known nanoviruses. The four DNA-R molecules were highly diverse, sharing only 65-71% identity with each other and 64-86% identity with those of other nanoviruses. In the S. alopecuroides plant 14 molecules sharing 57.7-84.6% identity with previously determined sequences of nanovirus-associated alphasatellites were also identified. Given the research activity in the nanovirus field during the last five years coupled with high-throughput sequence technologies, many more diverse nanoviruses and nanovirus-associated satellites are likely to be identified.

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

Implementation 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.

Nucleotide sequence analyses of coat protein gene of peanut stunt virus isolates from alfalfa and different hosts show a new tentative subgroup from Iran.

Alfalfa cultivars grown in 14 provinces in Iran were surveyed for the relative incidence of peanut stunt virus (PSV) during 2013-2016. PSV were detected in 41.89% of symptomatic alfalfa samples and a few alternate hosts by plate-trapped antigen ELISA. Among other hosts tested only Chenopodium album, Robinia pseudoacacia and Arachis hypogaea were found naturally infected with PSV. Twenty five isolates of PSV were chosen for biological and molecular characterizations based on their geographical distributions. There was not any differences in experimental host range of these isolates; however, variation in systemic symptoms observed on Nicotiana glutinosa. Total RNA from 25 of viral isolates were subjected to reverse transcription polymerase chain reaction analysis using primers directed against coat protein (CP) gene. The CP genes of 25 Iranian PSV isolates were either 651 or 666 nucleotides long. The nucleotide and amino acid identities for CP gene among Iranian PSV isolates were 79.3-99.7 and 72-100%, respectively. They also shared between 67.4 and 82.4% pairwise nucleotide identity with other PSV isolates reported elsewhere in the world. Phylogenetic analyses of CP gene sequences showed formation of a new subgroup comprising only the Iranian isolates. Natural infection of a few alternate hosts with PSV is reported for the first time from Iran.

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.

Molecular diversity of turncurtoviruses in Iran.

Turnip curly top virus (TCTV) is the only member of the newly established genus Turncurtovirus (family Geminiviridae). As part of an ongoing study to identify additional plant hosts and the diversity of turncurtoviruses, between 2012 and 2014, we sampled symptomatic turnip plants and other crops in the provinces Fars and Khorasan Razavi (southern and northeastern Iran, respectively). Infection by turncurtoviruses was tested by PCR and/or rolling-circle amplification (RCA) coupled with restriction enzyme digests. Turncurtoviruses were identified in turnip as well as seven other field crops, including eggplant, basil, radish, lettuce, sugar beet, red beet and spinach. Full turncurtovirus genomes were recovered from 25 of these samples, leading to the identification of TCTV and a new putative turncurtovirus, turnip leaf roll virus (TLRV; 13 isolates), which shares <80% genome-wide pairwise identity with TCTV. Agroinoculation of plants with an infectious clone of TLRV demonstrated that this virus could infect several plant hosts under greenhouse conditions and could be transmitted by the leafhopper Circulifer haematoceps (Mulsant and Rey, 1855) from agroinoculated to healthy plants.

Analysis of watermelon chlorotic stunt virus and tomato leaf curl Palampur virus mixed and pseudo-recombination infections.

Watermelon chlorotic stunt virus (WmCSV) and tomato leaf curl Palampur virus (ToLCPMV) are limiting factors for cucurbit production in south and southeastern Iran. ToLCPMV infects all cucurbit crops (except watermelons) whereas WmCSV is somewhat limited to watermelon, causing detrimental effects on fruit production. In a survey, we detected WmCSV in all watermelon growing farms in Fars province (southern Iran). Given that WmCSV and ToLCPMV are present in the same geographical location in Iran, we studied the interaction of two viruses. Co-infection using agroinfectious clones of WmCSV and ToLCPMV caused severe symptoms in watermelon and zucchini in comparison to symptoms observed from individual infections. Interestingly, inoculation of zucchini with WmCSV DNA-A and ToLCPMV DNA-B agroinfectious clones or vice versa produced a viable pseudo-recombinant and induced systemic symptoms. This demonstrates that replication-associated protein of DNA-A of each virus is able to bind to cis elements of the DNA-B molecules of another virus.

Incidence and characterization of Potato virus V infections in Iran.

A survey was carried out to determine the extent of Potato virus V (PVV) infection, together with other potyviruses, in Iran in both commercial and local potato varieties. We found a low incidence of PVV in commercial varieties compared to a local potato cultivar Zardi, in which we noted a PVV infection up to ~32.9 %. We determined the genomic sequence 9,812 nucleotides of one isolate (KER.LAL.P) from cultivar Agria and the 3'-terminal sequence including coat protein (CP) gene of four additional isolates from cultivar Zardi. The Iranian isolate PVV KER.LAL.P was found to share 91 % sequence identity with the Scottish isolate DV-42 (AJ243766) of PVV. The CP gene sequences of the PVV isolates from Iran shared 96.5 to 99.3 % pairwise nucleotide identity and they shared <97.5 % pairwise identity with the CP sequences of all other PVV isolates available in public databases. Our host studies indicated that the Iranian PVV isolate had a narrower host range and infected Nicotiana debneyi and N. glutinosa test plants. Within the commercial varieties of potato in Iran, we noted a significant amount of mixed potyvirus infection. This study is the first report of occurrence and complete genome of PVV in Iran.

Molecular characterization and field survey of Iranian potato virus X isolates.

Six hundred and one symptomatic potato samples were collected from nine provinces in Iran. Screening by double-antibody sandwich enzyme linked immunosorbent assay using a potato virus X (PVX) together with a few potyviruses polyclonal antibodies, produced positive reactions in 4.3 % of samples against PVX. Based on symptoms on different test plant, the isolates were divided into two groups: the first groups causing blistering and malformation of leaves and the second showed mild mosaic and vein clearing in Nicotiana glutinosa. The almost complete nucleotide sequence of two isolates as a representative of severe and a mild isolates were determined. Genomes of two PVX Iranian isolates are identical to that of the most PVX isolates comprise 6435 nucleotides in length excluding 101 nucleotide in the 5' end of the genome and shares 94.8-96.7 % identities with European and Asian, and 77-96.1 % with American isolates. Furthermore, the 3'-terminal sequences, including the coat protein coding region of other 13 Iranian isolates were determined and compared with the GenBank sequences. Phylogenetic analysis of the cp gene of 13 Iranian isolates together all those available in public databases indicated that the 13 Iranian isolates all belong to low diversity clade I.

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.

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.

Genetic diversity and host range studies of turnip curly top virus.

Turnip curly top virus (TCTV) is a unique geminivirus that has recently been characterised as infecting turnips in Iran. The genome of TCTV shares <68 % pairwise identity with other geminiviruses and has a genome organisation similar to that of curtoviruses and topocuvirus. The replication-associated protein (Rep) bears the highest similarity to curtovirus Reps (48.5-69.0 %); however, in the case of the capsid protein (CP), the extent of similarity is only 39.5-44.5 %. We constructed an agroinfectious clone of TCTV and undertook host range studies on ten plant species; in three species (turnip, sugar beet and cowpea), we detected infection which presents curly top symptoms in turnip and sugar beet. The efficiency of TCTV infection in agroinoculated turnip plants was 71.7 %, and the infection was successfully transmitted to 80 % of the healthy turnip plants used in the insect transmission studies by Circulifer haematoceps under greenhouse conditions. We also determined the genome sequence of 14 new TCTV isolates from southern Iran isolated from turnips. We observed ~13 % diversity amongst all the TCTV isolates and found evidence of recombination in the CP- and Rep-coding regions of the genomes.

Analysis of Iranian Potato virus S isolates.

Two hundred forty potato samples with one or more symptoms of leaf mosaic, distortion, mottling and yellowing were collected between 2005 and 2008 from seven Iranian provinces. Forty-four of these samples tested positive with double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA) using a Potato virus S (PVS) polyclonal antibody. Of these 12 isolates of PVS were selected based on the geographical location for biological and molecular characterization. The full coat protein (CP) and 11K genes from 12 PVS isolates were PCR amplified, cloned and sequenced. All 12 PVS isolates showed mosaic symptoms on Nicotiana debneyii and N. tabacum cv. Whiteburly and local lesion on Chenopodium amaranticolor, C. quinoa and C. album. The Iranian isolates share between 93 and 100% pairwise nucleotide identity with other PVS(O) isolates. Based on maximum likelihood phylogenetic analysis coupled with pairwise identity analysis, we propose 15 genotypes for the PVS(O) strain and 3 genotypes for the PVS(A) strain.

Characterisation of potato virus Y isolates from Iran.

A survey of Potato virus Y (PVY) was conducted in cultivated fields in six Iranian provinces between January 2005 to July 2007. Two hundred samples from potato and tomato were collected and analyzed by enzyme-linked immunosorbent assay (ELISA) for potyviruses. Almost one fourth of the samples were found to be infected by PVY. Analysis of these PVY-positive samples using three monoclonal antibodies (MAbs) facilitating the simultaneous detection of three main strains namely the ordinary (PVY(O)), strain (PVY(N)) and C (PVY(C)) strains. However, the fourth strain (PVY(NTN)) and some others recombinant isolates were also identified by molecular methods. Host range and symptoms analysis using sap inoculation of four different strains of PVY onto a range of plants revealed that the four strains showed biological properties that seemed to be consistent with their molecular grouping. Fourteen isolates of PVY were chosen based on the host and geographical location, primer specificity and serology for further biological and molecular characterisation. The coat protein (CP) and P1 genes and 3'-non-translated region (3'NTR) from 14 representative isolates were sequenced and analysed with the sequences available in GenBank. Composite analysis of the P1, CP and 3'-UTR sequences with all full genome sequences of PVY revealed that there are three potential strains of PVY in Iran, PVY(O), PVY(N)-W and PVY(NTN). Isolate KER.SA(N) was the most divergent of all the 14 isolates reacted with PVY(N) specific MAbs but grouped with PVY(O) strains in maximum likelihood phylogentic analysis. The PVY(NTN) isolates from Iran more closely related to the European than North American PVY(NTN) isolates.