Complete genome sequence of hollyhock vein yellowing virus, a novel monopartite begomovirus infecting hollyhock in Pakistan.

Hollyhock (Alcea rosea, family Malvaceae) is an ornamental plant grown widely in gardens across South Asia. In a bed of ornamental plants near the village of Chakri (Punjab Province, Pakistan) in 2014, hollyhock plants showing two distinct symptom types were identified: yellow vein mosaic and leaf crumple. PCR amplification with universal primers amplified a begomovirus from separate nucleic acid extracts of single plants of each type but amplified a betasatellite only from the plant with the yellow vein mosaic symptoms. No potential begomovirus DNA B component or alphasatellite could be identified in either sample. After cloning, the genome sequences of two viruses, one from a plant of each symptom type, were determined and shown to share 99.9% nucleotide sequence identity with each other but less than 91% nucleotide sequence identity with all previously characterized begomoviruses, with the highest identity (90%) to an isolate of pedilanthus leaf curl virus (PeLCV). This indicates that the two hollyhock plants were infected with a newly identified begomovirus for which the name "hollyhock vein yellowing virus" (HoVYV) is proposed. HoVYV likely has a recombinant origin. The betasatellite showed the highest nucleotide sequence identity to an isolate of cotton leaf curl Multan betasatellite (CLCuMuB), a betasatellite associated with cotton leaf curl disease across Pakistan and northwestern India. These findings add to the diversity of known begomoviruses in South Asia and again highlight the role of hollyhock as a reservoir of the cotton leaf curl begomovirus betasatellite complex. The results also suggest that the yellow vein mosaic symptoms in hollyhock are due to the betasatellite rather than the virus.

Complete genome sequence of a previously undescribed monopartite begomovirus and betasatellite infecting Malvastrum coromandelianum in Cambodia.

A previously undescribed monopartite begomovirus was identified in Kampot province, Cambodia, in Malvastrum coromandelianum plants exhibiting yellow vein symptoms characteristic of begomovirus infections. The apparently full-length viral component was cloned and sequenced following enrichment of circular DNA by rolling-circle amplification and restriction enzyme digestion. The genome of the virus was 2737 nucleotides in length (KP188831) and exhibited an organization like that of other monopartite begomoviruses, sharing the highest nucleotide sequence similarity (87.7% identity) with ageratum yellow vein virus (AM940137). A satellite molecule was amplified from total DNA by PCR amplification, using the betasatellite-specific primer pair ß01/ß02. The satellite molecule (1346 nt, KP188832) had structural characteristics like those of other betasatellites associated with begomoviruses and shared the highest nucleotide sequence similarity (84.8% identity) with malvastrum yellow vein betasatellite (MN205547). According to the criteria established for species demarcation for classification of begomoviruses (family Geminiviridae) and betasatellites (family Tolecusatellitidae), respectively, the virus isolate from M. coromandelianum in Cambodia is a previously undescribed novel monopartite begomovirus, for which the name "malvastrum yellow vein Cambodia virus" (MaYVCV) is proposed, and the betasatellite is a previously undescribed novel betasatellite, for which the name "malvastrum yellow vein Cambodia betasatellite" (MaYVKHB) is proposed.

Association of a begomovirus-satellite complex with yellow vein and leaf curl disease of hollyhock (Alcea rosea) in India.

Geminiviruses cause considerable yield loss in several crop plants worldwide. In 2016, several hollyhock plants displaying yellow mosaic and leaf curling symptoms were noticed in a nursery of Jawaharlal Nehru University, New Delhi, India. Analysis of the collected samples indicated an association of monopartite and bipartite begomoviruses with satellites. Three begomoviruses (including a member of a new begomovirus species), two alphasatellites, and a betasatellite were isolated from yellow-mosaic-disease-affected plants. Similarly, a begomovirus, two alphasatellites, and a betasatellite were found to be associated with leaf curl disease of hollyhock. These begomoviruses and satellites were found to be recombinants. By harboring diverse begomoviruses and satellite DNAs, hollyhock may serve as a potential source of virus inoculum.

Molecular characterization of two previously undescribed begomovirus-associated alphasatellite molecules infecting malvaceous species in Cameroon.

Two begomovirus-associated alphasatellites were isolated from okra and a malvastrum plant (Malvaceae) in Cameroon. The complete nucleotide sequences of the okra- and malvastrum-infecting alphasatellites were 1375 and 1416-1418 nucleotides, respectively, and both exhibited features characteristic of other alphasatellites. Based on pairwise sequence comparisons, these previously undescribed alphasatellites are members of distinct species in the genera Colecusatellite and Gosmusatellite and have been tentatively named "pepper yellow vein Mali alphasatellite" and "cotton leaf curl Gezira alphasatellite3", respectively. Taken together with previous studies, alphasatellites endemic to Cameroon appear to be more diverse and infect plants of many more species and families than currently recognized.

Two new begomoviruses that infect non-cultivated malvaceae in Brazil.

A high diversity of begomoviruses that infect non-cultivated plants has been noted in Brazil. Here, we report the complete sequences of two new species of bipartite begomoviruses from Sida sp. plants collected in the state of Piauí, northeastern Brazil. The genomes of these viruses show a genomic organization that is typical of New World begomoviruses. In phylogenetic analysis, two closely related viruses (sida angular mosaic virus, SiAMV and sida chlorotic vein virus, SiCVV) clustered with other begomoviruses described in tomato and Sida plants in Brazil. Evidence of recombination is shown among isolates of the species described.

Deciphering the biology of deltasatellites from the New World: maintenance by New World begomoviruses and whitefly transmission.

Deltasatellites are small noncoding DNA satellites associated with begomoviruses. The study presented here has investigated the biology of two deltasatellites found in wild malvaceous plants in the New World (NW). Infectious clones of two NW deltasatellites (from Malvastrum coromandelianum and Sidastrum micranthum) and associated begomoviruses were constructed. Infectivity in Nicotiana benthamiana and their natural malvaceous hosts was assessed. The NW deltasatellites were not able to spread autonomously in planta, whereas they were maintained by the associated bipartite begomovirus. Furthermore, NW deltasatellites were transreplicated by a monopartite NW begomovirus, tomato leaf deformation virus. However, they were not maintained by begomoviruses from the Old World (tomato yellow leaf curl virus, tomato yellow leaf curl Sardinia virus and African cassava mosaic virus) or a curtovirus (beet curly top virus). NW deltasatellites did not affect the symptoms induced by the helper viruses but in some cases reduced their accumulation. Moreover, one NW deltasatellite was shown to be transmitted by the whitefly Bemisia tabaci, the vector of its helper begomoviruses. These results confirm that these molecules are true satellites. The availability of infectious clones and the observation that NW deltasatellites reduced virus accumulation paves the way for further studies of the effect on their helper begomoviruses.

Complete nucleotide sequence of a new begomovirus infecting a malvaceous weed in Brazil.

Begomoviruses are single-strand DNA plant viruses that infect economically important crops worldwide, exhibiting high genetic variability and species diversity. Based on the current taxonomic criteria established for the genus Begomovirus, a new member of this genus infecting a malvaceous weed is reported here. The name triumfetta yellow mosaic virus is proposed. At least one recombination event was detected in this new begomovirus, with putative parents being begomoviruses from tomato and Centrosema.

Complete nucleotide sequences of a new bipartite begomovirus from Malvastrum sp. plants with bright yellow mosaic symptoms in South Texas.

Two isolates of a novel bipartite begomovirus, tentatively named malvastrum bright yellow mosaic virus (MaBYMV), were molecularly characterized from naturally infected plants of the genus Malvastrum showing bright yellow mosaic disease symptoms in South Texas. Six complete DNA-A and five DNA-B genome sequences of MaBYMV obtained from the isolates ranged in length from 2,608 to 2,609 nucleotides (nt) and 2,578 to 2,605 nt, respectively. Both genome segments shared a 178- to 180-nt common region. In pairwise comparisons, the complete DNA-A and DNA-B sequences of MaBYMV were most similar (87-88 % and 79-81 % identity, respectively) and phylogenetically related to the corresponding sequences of sida mosaic Sinaloa virus-[MX-Gua-06]. Further analysis revealed that MaBYMV is a putative recombinant virus, thus supporting the notion that malvaceous hosts may be influencing the evolution of several begomoviruses. The design of new diagnostic primers enabled the detection of MaBYMV in cohorts of Bemisia tabaci collected from symptomatic Malvastrum sp. plants, thus implicating whiteflies as potential vectors of the virus.

Novel begomoviruses recovered from Pavonia sp. in Brazil.

Begomoviruses are whitefly-transmitted, single-stranded DNA viruses that cause serious infections in crop plants and are often also associated with non-cultivated plants. Here, we report the detection of two new begomoviruses in Pavonia sp. (Malvaceae). Sequence comparisons and phylogenetic analysis showed that these novel viruses are related to New World begomoviruses. The nucleotide sequences of the DNA-A of both viruses had the highest similarity to abutilon mosaic Bolivia virus (AbMBoV). Based on symptoms observed in the field and considering the host, we propose the names pavonia mosaic virus (PavMV) and pavonia yellow mosaic virus (PavYMV) for these two new begomoviruses.

Complete nucleotide sequences of two new begomoviruses infecting the wild malvaceous plant Melochia sp. in Brazil.

Wild malvaceous plants are hosts for a large number of begomoviruses (genus Begomovirus, family Geminiviridae) in both the Old World and the New World. Here, we report the complete genome sequences of two new begomoviruses from Melochia sp. plants from Brazil. The cloned bipartite genomes, composed of DNA-A and DNA-B, showed the typical organization of the New World begomoviruses but they were distantly related to the genomes of other begomoviruses. We propose the names Melochia mosaic virus and Melochia yellow mosaic virus for these begomoviruses.

High genetic homogeneity points to a single introduction event responsible for invasion of Cotton leaf curl Multan virus and its associated betasatellite into China.

BACKGROUND: Cotton leaf curl Multan virus (CLCuMuV) is a Whitefly Transmitted Geminivirus (WTG) endemic to the India subcontinent and is notorious as a causal agent of cotton leaf curl disease (CLCuD), a major constraint to cotton production in south Asia. We found CLCuMuV infecting Hibiscus rosa-sinensis in Guangzhou, China in 2006. The spread and evolution of the invading CLCuMuV were monitored in the following nine years. FINDINGS: CLCuMuV spread rapidly in the last nine years and became established in Southern China. It infects at least five malvaceous plant species, H. rosa-sinensis, H. esculentus, Malvaiscus arboreus, Gossypium hirsutum and H. cannabinus. Complete nucleotide sequences of 34 geographically and/or temporally distinct CLCuMuV isolates were determined and analyzed together with six other publicly available genomes of CLCuMuV occurring in China. The 40 CLCuMuV isolates were found to share > 99 % nucleotide sequence identity with each other. In all cases tested, the CLCuMuVs were associated with a CLCuMuB. The 36 CLCuMuBs (30 sequenced by us) shared > 98 % nucleotide sequence identity. CONCLUSION: The high genetic homogeneity of CLCuMuV and CLCuMuB in China suggests the establishment of them from a single founder event.

Evolutionary liberties of the Abutilon mosaic virus cluster.

Two new strains of Abutilon mosaic virus (AbMV; Geminiviridae) from Germany (Stuttgart) and France (Paris) have been characterized by circomics, direct pyrosequencing of rolling circle amplification (RCA) products, as well as conventional cloning and Sanger sequencing. RCA combined with an analysis of restriction fragment length polymorphisms confirmed the completeness of the sequence determination and a close relationship of both isolates for DNA A with 99 % nucleotide sequence identity. Phylogenetic tree reconstruction supported their clustering with other AbMV strains in a clade with Middle American begomoviruses, whereas South American begomoviruses that infect Abutilon or Sida micrantha are less closely related. Comparing the coat protein (CP) genes of the AbMV cluster, with those of related Middle and South American begomoviruses revealed a remarkable overrepresentation for non-synonymous nucleotide exchanges for certain amino acid positions in the AbMV cluster. Projection of these positions to a structural model of the African cassava mosaic virus CP yielded a non-random distribution at the periphery and, most importantly, highlighted those amino acids that had been identified in whitefly-transmission experiments before. These results establish the basis for an analysis of the evolutionary liberty of certain amino acid positions of the CP, and their impact on the deciphering of insect transmission determinants is discussed.

Association of a distinct strain of hollyhock yellow vein mosaic virus and Ludwigia leaf distortion betasatellite with yellow vein mosaic disease of hollyhock (Alcea rosea) in India.

A distinct strain of hollyhock yellow vein mosaic virus (HoYVMV) and Ludwigia leaf distortion betasatellite (LuLDB) were associated with yellow vein mosaic of hollyhock. The viral DNA genome (JQ911766) and betasatellite (JQ408216) shared highest nucleotide sequence identity (89.2 %) with HoYVMV (the only available sequence in GenBank) and 92 % identity with LuLDB. Agroinfiltration of HoYVMV and LuLDB induced yellow vein mosaic symptoms on hollyhock, thereby demonstrating causality of the disease.

A novel begomovirus isolated from sida contains putative cis- and trans-acting replication specificity determinants that have evolved independently in several geographical lineages.

A novel begomovirus isolated from a Sida rhombifolia plant collected in Sinaloa, Mexico, was characterized. The genomic components of sida mosaic Sinaloa virus (SiMSinV) shared highest sequence identity with DNA-A and DNA-B components of chino del tomate virus (CdTV), suggesting a vertical evolutionary relationship between these viruses. However, recombination analysis indicated that a short segment of SiMSinV DNA-A encompassing the plus-strand replication origin and the 5´-proximal 43 codons of the Rep gene was derived from tomato mottle Taino virus (ToMoTV). Accordingly, the putative cis- and trans-acting replication specificity determinants of SiMSinV were identical to those of ToMoTV but differed from those of CdTV. Modeling of the SiMSinV and CdTV Rep proteins revealed significant differences in the region comprising the small ß1/ß5 sheet element, where five putative DNA-binding specificity determinants (SPDs) of Rep (i.e., amino acid residues 5, 8, 10, 69 and 71) were previously identified. Computer-assisted searches of public databases led to identification of 33 begomoviruses from three continents encoding proteins with SPDs identical to those of the Rep encoded by SiMSinV. Sequence analysis of the replication origins demonstrated that all 33 begomoviruses harbor potential Rep-binding sites identical to those of SiMSinV. These data support the hypothesis that the Rep ß1/ß5 sheet region determines specificity of this protein for DNA replication origin sequences.

Mixed infection of Sida jamaicensis in Jamaica reveals the presence of three recombinant begomovirus DNA A components.

Begomoviruses impose serious constraints on agriculture throughout the temperate, tropical and subtropical regions. Previously, we characterised a sida golden yellow vein virus isolate, SiGYVV-[JM:Lig2:08] (HQ009519-20) from a symptomatic Sida jamaicensis plant. With the aim of establishing whether it was hosting a mixed infection that could facilitate recombination, PCR-RFLP was done on DNA extracted from this plant, and the results suggested the presence of two additional genetically distinct DNA-A molecules. Sequence analysis of these two DNA-A molecules (relying on BLAST searches and the CLUSTAL V algorithm within the DNASTAR MegAlign module) revealed that they belonged to novel species, and we have tentatively named these viruses sida golden mosaic Braco virus-[Jamaica:Liguanea:2008] and sida golden mosaic Liguanea virus-[Jamaica:1:2008]. Using RDP4 (recombination detection program), we determined that all three viruses were recombinant, with bases ~10 to ~440 of both SiGMLigV-[JM:Lig:08] and SiGYVV-[JM:Lig2:08] having been derived from a relative of SiGMBV-[JM:Lig:08] (P<2.070×10(-7) for all seven of the recombination detection methods). SiGMBV-[JM:Lig:08] was itself a product of recombination, deriving bases ~490-1195 from a virus that was ~92% similar to malvastrum yellow mosaic Helshire virus. Phylogenetically, these DNA-A components are most closely related to those of malvaceous weed-infecting begomoviruses from Jamaica, Cuba, Florida and Mexico. The SiGMBV DNA-A was able to elicit symptomatic infection in N. benthamiana.

The molecular characterisation of a Sida-infecting begomovirus from Jamaica.

The complete DNA sequence of both genome components of a new begomovirus (Sida golden mosaic Buckup virus-[Jamaica:St. Elizabeth:2004]; SiGMBuV-[JM:SE:04]) was determined from a field-infected Sida sp. sample from Buckup, St. Elizabeth, Jamaica. Phylogenetically, both genome components of SiGMBuV-[JM:SE:04] are most closely related to malvaceous weed-infecting Floridian and Mexican begomoviruses. Its DNA-B is a recombinant molecule, the majority of which was derived from a virus resembling Sida yellow mosaic Yucatan virus-[Mexico:Yucatan:2005] (SiYMYuV-[MX:Yuc:05]), while nucleotides 43-342 were derived from a virus resembling Sida golden mosaic virus-[United States of America:Florida] (SiGMV-[US:Flo]). Symptomatic infectivity of our cloned SiGMBuV-[JM:SE:04] components was confirmed in Nicotiana benthamiana.

Molecular and biological characterization of corchorus mottle virus, a new begomovirus from Brazil.

A begomovirus infecting Orinoco jute (Corchorus hirtus) from Brazil was characterized. Molecular analysis revealed a bipartite genomic organization, which is typical of the New World begomoviruses. Sequence analysis and phylogenetic data showed that both genomic components have the closest relationship with abutilon mosaic Brazil virus, with an identity of 87.3 % for DNA-A, indicating that this virus is a member of a new begomovirus species for which the name "Corchorus mottle virus" (CoMoV) is proposed. Sida rhombifolia plants inoculated by biolistics with an infectious clone of CoMoV showed systemic vein chlorosis, mottling and leaf deformation symptoms, while Nicotiana benthamiana and tomato plants had symptomless infection. CoMoV is the first corchorus-infecting begomovirus reported in Brazil.

A recombinant begomovirus resulting from exchange of the C4 gene.

A begomovirus isolated from Malvastrum coromandelianum and tomato originating from Yunnan province (China) was shown to be representative of a new begomovirus species, for which the name tomato leaf curl Yunnan virus (TLCYnV) is proposed. TLCYnV has high levels of sequence identity to tomato yellow leaf curl China virus (TYLCCNV) across the whole genome, except for sequences encompassing the C4 gene. Agrobacterium-mediated inoculation showed TLCYnV to be highly infectious to a range of plant species but poorly infectious to M. coromandelianum. In contrast to TYLCCNV, TLCYnV was shown to infect tomato in the absence of a betasatellite. In field-collected samples, TLCYnV was identified most frequently in tomato in which it was not associated with a betasatellite. Transgenic expression in Nicotiana benthamiana showed that the C4 protein of TYLCCNV did not induce developmental abnormalities, whereas the C4 of TLCYnV induced severe developmental abnormalities, reminiscent of virus symptoms. The genome of TLCYnV was shown to be significantly less methylated in plants than that of TYLCCNV and the C4 protein of TLCYnV was shown to suppress post-transcriptional gene silencing and transcriptional gene silencing more effectively than the C4 of TYLCCNV. The results indicate that TLCYnV evolved from TYLCCNV by recombination, acquiring a more virulent C4, allowing it to dispense with the requirement for a betasatellite. The implications of these findings in relation to the evolution of monopartite begomoviruses are discussed.

A study of weeds as potential inoculum sources for a tomato-infecting begomovirus in central Brazil.

Tomato severe rugose virus (ToSRV) is the most important begomovirus species in Brazilian tomato production. Many weeds are associated with tomato, and some are hosts of begomoviruses. Only one species of weed, Nicandra physaloides, has been found to be infected with ToSRV. In this study, four weed species were investigated for their capacity to be infected by ToSRV and serve as a potential source of inoculum for tomato. Begomoviruses from naturally infected Crotalaria spp., Euphorbia heterophylla, N. physaloides, and Sida spp. were successfully transferred to tomato plants by biolistic inoculation. ToSRV was the major virus transferred to tomato. In contrast, other begomoviruses were transferred to weeds, such as Sida micrantha mosaic virus and Euphorbia yellow mosaic virus. Furthermore, a new strain of Sida micrantha mosaic virus is reported. We also confirmed that Crotalaria spp., E. heterophylla, and Sida spp. are infected with ToSRV but at low viral titers and in mixed infections with weed-infecting begomoviruses. Thus, it was demonstrated that weeds are potential sources of ToSRV for tomato in central Brazil.

Characterization of sequence elements from Malvastrum yellow vein betasatellite regulating promoter activity and DNA replication.

BACKGROUND: Many monopartite begomoviruses are associated with betasatellites, but only several promoters from which were isolated and studied. In this study, the ßC1 promoter from Malvastrum yellow vein betasatellite (MYVB) was characterized and important sequence elements were identified to modulate promoter activity and replication of MYVB. RESULTS: A 991 nucleotide (nt) fragment upstream of the translation start site of the ßC1 open reading frame of MYVB and a series of deletions within this fragment were constructed and fused to the ß-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes, respectively. Agrobacterium-mediated transient expression assays showed that the 991 nt fragment was functional and that a 28 nt region (between -390 nt and -418 nt), which includes a 5'UTR Py-rich stretch motif, was important for promoter activity. Replication assays using Nicotiana benthamiana leaf discs and whole plants showed that deletion of the 5'UTR Py-rich stretch impaired viral satellite replication in the presence of the helper virus. Transgenic assays demonstrated that the 991 nt fragment conferred a constitutive expression pattern in transgenic tobacco plants and that a 214 nt fragment at the 3'-end of this sequence was sufficient to drive this expression pattern. CONCLUSION: Our results showed that the ßC1 promoter of MYVB displayed a constitutive expression pattern and a 5'UTR Py-rich stretch motif regulated both ßC1 promoter activity and MYVB replication.