DNAβ Molecule Research Articles

Complete nucleotide sequence of Croton yellow vein mosaic virus and DNA-β associated with yellow vein mosaic disease of Jatropha gossypifolia in India

A severe yellow vein mosaic disease was noticed on several Jatropha gossypifolia plants growing nearby agriculture fields at Lucknow, India. Diseased plants exhibited yellow vein mosaic, leaf deformation, vein swelling and stunting. A population of whiteflies (Bemisia tabaci) was also noticed in the vicinities; therefore, begomovirus infection was suspected. To confirm begomovirus association, total DNA was isolated from symptomatic leaf samples and subjected to PCR using DNA-A, DNA-B and DNA-β-specific primers. DNA-A and DNA-β was successfully amplified but several attempts failed to amplify DNA-B indicating monopartite nature of the begomovirus. The sequence analysis of amplicons revealed the presence of 2757 nucleotides of DNA-A genome (EU727086) and 1315 nt of DNA-β molecule (EU604296). The sequence analysis of DNA-A (EU727086) revealed the highest 96% identities and closest relationship with Croton yellow vein mosaic virus (CYVMV, AJ507777) infecting Croton bonplandianum in India. The DNA-β (EU604296) showed the highest 96% sequence identity and closest phylogenetic relationship with CYVMV-associated DNA-β (AM410551) isolated from Croton sp. in Pakistan. Based on the highest sequence identities and closest phylogenetic relationships of the DNA-A genome and DNA-β molecule with respective sequences of various isolates of Croton yellow vein mosaic virus, the begomovirus associated with yellow vein mosaic disease of J. gossypifolia was identified as an isolate of Croton yellow vein mosaic virus.

A novel recombinant tomato-infecting begomovirus capable of transcomplementing heterologous DNA-B components

The genome of a tomato-infecting begomovirus from Ranchi, India, was cloned, sequenced and analysed. The viral genome shared 88.3% sequence identity with an isolate belonging to the species Tobacco curly shoot virus (TbCSV), and this virus should therefore be considered a member of a new species, tentatively named Tomato leaf curl Ranchi virus (ToLCRnV). The DNA-β molecule, which had 74.5% sequence identity with tomato leaf curl Bangladesh betasatellite (ToLCBDB), is named tomato leaf curl Ranchi betasatellite (ToLCRnB). Phylogenetic analysis revealed that ToLCRnV is related to tomato leaf curl Bangladesh virus (ToLCBDV), tobacco curly shoot virus (TbCSV) and tomato leaf curl Gujarat virus (ToLCGV). An infectivity study with ToLCRnV established the monopartite nature of the viral genome, whereas inoculation with ToLCRnB resulted in increased symptom severity. ToLCRnV could transreplicate DNA-B of tomato leaf curl Gujarat virus (ToLCGV) and tomato leaf curl New Delhi virus (ToLCNDV), both in N. benthamiana and tomato, although DNA-B accumulation of was less than with the wild-type combinations. ToLCRnB could be efficiently replicated by DNA-A of both ToLCNDV and ToLCGV. A leaf disk assay suggests that DNA-A could transreplicate the homologous DNA-B and DNA-β more efficiently than the heterologous one.

Molecular characterization of a new strain of tomato leaf curl Philippines virus and its associated satellite DNAβ molecule: further evidence for natural recombination amongst begomoviruses

Summary The full-length genome of a begomovirus and its cognate DNAb component associated with tomato leaf curl disease (ToLCD), from Laguna, the Philippines, were cloned. Sequence analysis revealed that the viral genome (AB307731) is 2,746 bp and the DNAb satellite (AB307732) is 1,346 bp in length. The genome is homologous to the DNA-A of monopartite begomoviruses originating from the Old World, with six conserved ORFs. Further nucleotide (nts) sequence comparisons showed that the genome has the highest sequence identities (94%) with that of tomato leaf curl Philippines virus-Los Banos (ToLCPV-LB). The DNAb satellite shared relatively low sequence identity (59–68%) with other DNAb satellite molecules. Recombination analysis indicated that ToLCPV-Laguna probably originated from another begomoviruses related to ToLCPV-Los Banos:L3, as a major parent, and ageratum yellow vein Hualian virus[Taiwan:Hsinchu:tom:2003] (AYVHV-[TW:His:tom:2003]) as minor parent. In this recombination event, our results revealed a high probability (RDP P value = 6.779 9 10 -9 ) that ToLCPV-Laguna has a chimeric genome, with nts 2,515 to 31 originating from ToLCPV-LB:L3 and the other part

Tomato yellow leaf curl virus and Tomato leaf curl Taiwan virus Invade South‐east Coast of China

AbstractAn epidemic outbreak of severe yellow leaf curl disease was reported in field grown tomato within Zhejiang Province of China in the autumn–winter cropping season of 2006. A molecular diagnostic survey was carried out based on comparisons of partial and complete viral DNA sequences. Comparison of partial DNA‐A sequences amplified with degenerate primers specific for begomoviruses confirmed the presence of two types of begomoviruses. The complete DNA sequences of five isolates, corresponding to the two types, were determined. Sequence comparisons and phylogenetic analysis revealed that they correspond to two previously identified begomoviruses, Tomato yellow leaf curl virus and Tomato leaf curl Taiwan virus. The satellite DNAβ molecule was not detected in these samples by either PCR or Southern blot hybridization analysis. There has been no previous report of geminivirus disease incidence in Zhejiang Province, indicating that the introduction of these two tomato infecting geminiviruses into the agro‐ecological zone of South‐eastern China is a fairly recent event. The implications for disease control are discussed.

Molecular Variation of Satellite DNAβ Molecules Associated with Malvastrum yellow vein virus and Their Role in Pathogenicity

Previous studies have found that the diversity of begomovirus-associated DNA beta satellites is related to host and geographical origin. In this study, we have cloned and sequenced 20 different isolates of DNA beta molecules associated with Malvastrum yellow vein virus (MYVV) isolated from Malvastrum coromandelianum plants in different geographical locations of Yunnan Province, China. Analyses of their molecular variation indicate that the satellites are clustered together according to their geographical location but that they have only limited sequence diversity. Infectivity tests using infectious clones of MYVV and its associated DNA beta molecule indicate that MYVV DNA beta is indispensable for symptom induction in Nicotiana benthamiana, N. glutinosa, Petunia hybrida, and M. coromandelianum plants. Furthermore, we showed that MYVV interacts functionally with heterologous DNA beta molecules in N. benthamiana plants.

First Report of Ageratum yellow vein virus Causing Tobacco Leaf Curl Disease in Fujian Province, China.

A leaf curling disease was observed on 7% of tobacco plants during December 2005 in research plots in the Cangshan District of Fuzhou, Fujian, China. Tobacco plants were infested with Bemisia tabaci, suggesting begomovirus etiology. To identify possible begomoviruses, total DNA was extracted from four symptomatic leaf samples (F1, F2, F3, and F4). The degenerate primers PA and PB were used to amplify part of the intergenic region and AV2 gene of DNA-A-like molecules (3). A 500-bp DNA fragment was amplified by PCR from all four samples. The PCR products were cloned and sequenced (GenBank Accession Nos. EF531601-EF531603 and EF527823). Alignment of the 500-bp sequences for the four isolates indicated that they shared 98.5 to 99.6% nt identity, suggesting that the plants were all infected by the same virus. Overlapping primers TV-Full-F (5'-GGATCCTCTTTTGAACGAGTTTCC-3') and TV-Full-R (5'-GGATCCCACATGTTTAAAATAATAC-3') were then designed to amplify the full-length DNA-A from sample F2. The sequence was 2,754 nucleotides long (GenBank Accession No. EF527823). A comparison with other begomoviruses indicated the F2 DNA-A had the highest nucleotide sequence identity (95.7%) with Ageratum yellow vein virus (AYVV; GenBank Accession No. X74516) from Singapore. To further test whether DNAβ was associated with the four viral isolates, a universal DNAβ primer pair (beta 01 and beta 02) was used (4). An amplicon of approximately 1.3 kb was obtained from all samples. The DNAβ molecule from F2 was then cloned and sequenced. F2 DNAβ was 1,345 nucleotides long (GenBank Accession No. EF527824), sharing the highest nucleotide sequence identity with the DNAβ of Tomato leaf curl virus (97.2%) from Taiwan (GenBank Accession No. AJ542495) and AYVV (88.8%) from Singapore (GenBank Accession No. AJ252072). The disease agent was transmitted to Nicotiana tabacum, N. glutinosa, Ageratum conyzoides, Oxalis corymbosa, and Phyllanthus urinaria plants by whiteflies (B. tabaci) when field infected virus isolate F2 was used as inoculum. In N. tabacum and N. glutinosa plants, yellow vein symptoms were initially observed in young leaves. However, these symptoms disappeared later during infection and vein swelling and downward leaf curling symptoms in N. tabacum and vein swelling and upward leaf curling in N. glutinosa were observed. In A. conyzoides, O. corymbosa, and P. urinaria plants, typical yellow vein symptoms were observed. The presence of the virus and DNAβ in symptomatic plants was verified by PCR with primer pairs TV-Full-F/TV-Full-R and beta 01/beta 02, respectively. The above sequence and whitefly transmission results confirmed that the tobacco samples were infected by AYVV. In China, Tobacco leaf curl Yunnan virus, Tobacco curly shoot virus, and Tomato yellow leaf curl China virus were reported to be associated with tobacco leaf curl disease (1,3). To our knowledge, this is the first report of AYVV infecting tobacco in China. A. conyzoides is a widely distributed weed in south China and AYVV was reported in A. conyzoides in Hainan Island, China (2). Therefore, this virus may pose a serious threat to tobacco production in south China.

Malvastrum leaf curl Guangdong virus is a distinct monopartite begomovirus

Virus isolates GD6, GD7, GD8, GD9 and GD10 were obtained from Malvastrum coromandelianum showing leaf curl symptoms in Guangdong Province of China. A specific 500 bp product was consistently detected in total DNA extracts, amplified with universal primers specific for members of the genus Begomovirus. Analysis of their partial DNA sequences revealed that they are isolates of the same begomovirus species, sharing 92·8%–97·1% nucleotide sequence identity. The complete DNA sequences of both GD6 and GD9 were found to be 2767 nucleotides, with all the characteristic features of begomovirus genome organization. The two isolates have less than 85·2% nucleotide sequence identity with other reported begomoviruses. Consequently, GD6 and GD9 are considered to be isolates of a novel begomovirus species, for which the name Malvastrum leaf curl Guangdong virus (MLCuGdV) is proposed. Sequence analyses suggest that MLCuGdV may have arisen by recombination between viruses related to Papaya leaf curl China virus, Tomato leaf curl Philippines virus and other undiscovered virus ancestors. Neither the DNA‐B component nor the DNAβ molecule associated with these begomovirus isolates was found. An infectious clone of GD6 was constructed. GD6 efficiently infected Nicotiana benthamiana, N. glutinosa and Petunia hybrida by agro‐inoculation, and Malvastrum coromandelianum by whitefly transmission, inducing leaf curling, vein swelling and stunting symptoms. GD6 was also infectious in N. tabacum, but did not induce observable disease symptoms.

Ageratum yellow vein China virus Is a Distinct Begomovirus Species Associated with a DNAβ Molecule

ABSTRACT Ageratum conyzoides plants exhibiting yellow vein symptoms, collected near Haikou, Hainan Province, China, contained begomoviral DNA-A-like molecules. The complete sequences of the molecules from two samples, Hn2 and Hn2-19, were shown to consist of 2,768 and 2,748 nucelotides (nt), respectively. These sequences have more than 97% nucleotide sequence identity, but less than 86% identity with other reported begomovirus sequences. In line with the taxonomic convention for begomoviruses, Hn2 and Hn2-19 are therefore considered to represent isolates of a distinct begomovirus species, for which the name Ageratum yellow vein China virus (AYVCNV) is proposed. Sequence alignment shows AYVCNV has arisen by recombination among viruses related to Ageratum yellow vein virus, Papaya leaf curl China virus, and an unidentified begomovirus. Southern blot analyses revealed that all plants sampled contained molecules resembling DNAbeta. DNAbeta molecules from three samples were 1,323 or 1,324 nt long and had >98% sequence identity but <81% identity with previously reported DNAbeta sequences. Infectious clones of Hn2 and its associated DNAbeta were constructed and agroinoculated to plants. Hn2 alone caused sporadic asymptomatic systemic infection of Nicotiana benthamiana, N. glutinosa, Lycopersicon esculentum, Petunia hybrida, and A. conyzoides but its accumulation was much enhanced in plants co-inoculated with DNAbeta. The co-inoculated N. benthamiana, N. glutinosa, P. hybrida, and L. esculentum plants developed leaf curling or leaf crinkling symptom; those in A. conyzoides were typical of ageratum yellow vein disease. When the DNAbeta molecules associated with four other Chinese begomoviruses were coinoculated with Hn2 to N. benthamiana and N. glutinosa, the DNAbeta molecules were replicated, and the plants developed systemic symptoms of types that were specific for each DNAbeta. This illustrates that there is less specific interaction between monopartite begomovirus and DNAbeta than between the DNA-A and DNA-B of begomoviruses with bipartite genomes.

Complete nucleotide sequences of Malvastrum yellow mosaic virus and its associated DNAβ molecule

Complete nucleotide sequences of Malvastrum yellow mosaic virus and its associated DNAβ molecule

Molecular Characterization of two Distinct Begomoviruses from Ageratum conyzoides and Malvastrum coromandelianum in China

AbstractTwo weed samples, G52 from Ageratum conyzoides and G87 from Malvastrum coromandelianum, showing leaf curling and vein thickening symptoms were collected in Nanning, Guangxi Province, China. The complete nucleotide sequences of DNA‐A‐like molecules of G52 and G87 were determined to be 2735 and 2745 nucleotides respectively. Both DNA‐A molecules have a genomic organization typical of begomoviruses and share 73.4% sequence identity with each other. Sequence comparisons showed that the DNA‐A of G52 and G87 were most closely related to those of Ageratum yellow vein virus (AYVV; 85% sequence identity) and Tobacco leaf curl Yunnanvirus (75.7% sequence identity) respectively. Further sequence comparisons showed that G52 has arisen by recombination among viruses related to AYVV, Papaya leaf curl China virus and an unidentified Begomovirus species. The molecular data suggest that G52 and G87 are two distinct begomoviruses, for which the names Ageratum leaf curl virus for G52 and Malvastrum leaf curl virus for G87 are proposed. The satellite DNAβ molecule was only found to be associated with G87. G87 DNAβ consists of 1354 nucleotides, and shares the highest nucleotide sequence identity (68.9%) with that associated with Sida yellow vein China virus. A defective DNAβ molecule was also found to be associated with G87.

A novel begomovirus with distinct genomic and phenotypic features infects tomato in Bangladesh

16 000 ha annually.Tomato leaf curl virus disease (ToLCVD) normally reduces tomato pro-duction significantly, often causing up to 100% yield loss. Diseased tomatoplants were collected from Joydebpur, Bangladesh in September 2003because they exhibited unusually mild ToLCVD symptoms of yellowingand leaf curling. In addition, mosaic-like symptoms developed on the olderleaves of susceptible tomato cv. Moneymaker, which is atypical for leafcurl-inducing viruses.Total DNA was extracted from leaves with symptoms and putative viralgenomes were amplified by PCR using degenerate primers specific to DNA-A and -B (Muniyappa et al., 2003), and DNA-β molecules (Briddon et al.,2002). Sequences generated from the amplicons were used either to designvirus-specific primers, or to obtain directly complete viral sequences. Apreviously undescribed monopartite begomovirus, designated Tomatoleaf curl Joydebpur virus-Mild (ToLCJV-Mld), was found associated withthese mild symptoms. Based on DNA-A sequences (2761 nucleotides,EMBL accession no. AJ875159), ToLCJV-Mld was most similar to Pepperleaf curl Bangladesh virus (AF314531) at 87·1% nucleotide (nt) identity.ToLCJV-Mld satellite DNA-β molecule (1366 nt, AJ966244) sharedhighest (59·0%) nt identity with a molecule isolated from ToLCVD inIndia (AY438558). No DNA-B component was detected. The number andarrangement of ToLCJV-Mld DNA-A open reading frames (ORFs) wasidentical to monopartite begomoviruses, except that an additional ORF of849 nt (282 amino acids) was predicted to occur on the complementarystrand between nt positions 1139 and 291, encompassing the entirelength of the coat protein gene. The association of a DNA-β moleculewith begomoviruses is known to cause severe symptoms, but this is the firstreport where it is associated with mild symptoms. ToLCJV-Mld DNA-Ashared 86·0 and 74·4% nucleotide identities with the previously reportedTomato leaf curl Bangladesh virus (AF188481) (Green et al., 2001) andTomato leaf curl New Delhi virus-Severe[Jessore] (AJ875157) (Maruthi etal., 2005), respectively. These results confirm the occurrence of at leastthree begomovirus species in tomato in Bangladesh.ReferencesBriddon RW, Bull SE, Mansoor S, Amin I, Markham PG, 2002. Universal primers for the PCR-mediated amplification of DNA- β : a molecule associated with monopartite begomoviruses.

Molecular Characterization of DNAβ Molecules Associated with Tobacco Leaf Curl Yunnan Virus*

Abstract Tobacco leaf curl Yunnan virus (TbLCYNV) isolates Y136 and Y143 were both shown to contain DNAβ molecules. Seven DNAβ molecules, each consisting of 1334–1341 nucleotides, were cloned and characterized. Sequence comparison showed that they were of two main types: type 1 included five DNAβ molecules with 75–99% overall nucleotide sequence identity, and type 2 included two DNAβ molecules with &gt;99% overall nucleotide sequence identity. Only 52–54% nucleotide identity was found between the two types of DNAβ molecules. The two isolates were associated with both types of DNAβ. A relationship dendrogram analysis showed that type 1 DNAβ molecules clustered with the DNAβ molecules of Tomato yellow leaf curl China virus, whereas type 2 DNAβ molecules clustered with the DNAβ of Tomato yellow leaf curl Thailand virus. Evidence was also provided that type 2 DNAβ molecules may have arisen by recombination between the DNAβ of Tobacco curly shoot virus and another unidentified virus. To date, only a single type of DNAβ molecule had been found to be associated with one begomovirus. The possible reasons of TbLCYNV associated with two types of DNAβ molecules are discussed.

Malvastrum yellow vein virus, a newBegomovirus species associated with satellite DNA molecule

Virus isolate Y47 was obtained fromMalvastrum coromandelianum showing yellow vein symptom in Honghe, Yunnan Province. The complete nucleotide sequence of DNA-A was determined, it contains 2731 nucleotides, having typical genomic organization of a begomovirus, encoding 6 ORFs with 2 ORFs [AV1(CP) and AV2] in virionsense DNA and 4 ORFs (AC1–AC4) in complementarysense DNA. Comparisons show that the total DNA-A of Y47 has the highest sequence identity (77%) with that ofOkra yellow vein mosaic virus- [201] (AJ002451), while less than 76% identities are found when compared with other begomoviruses. The molecular data show that virus isolate Y47 is a distinct begomovirus species, for which the nameMalvastrum yellow vein virus is proposed. Satellite DNA molecule (Y47β) was found to be associated with Y47 using the primers (beta01 and beta02) specific for DNAβ. Y47β consists of 1348 nucleotides, with a functional ORF (C1) in complementary-sense DNA. Y47β has 62%–67% sequence identity with DNAβ molecule associated withCotton leaf curl Multan virus orCotton leaf curl Rajasthan virus, while lower than 46% sequence identities are found when compared with other reported DNAβ molecules. Relationship dendrograms show that DNAβ molecules are co-evolved with their help begomoviruses.

Identification of a novel DNA molecule associated with To-bacco leaf curl virus

A circular DNA molecule, designated as DNAβ, was identified in tobacco plants infected with Tobacco leaf curl virus (TLCV) isolates Y5 and Y8 by PCR using primers based on the conserved region of the two reported DNAβ sequences of whitefly-transmitted geminiviruses (WTGs). The complete nucleotide sequences of DNAβ of Y5 and Y8 (TLCV DNAβ) were determined. Y5 DNAβ comprises 1333 nucleotides encoding 8 predicted ORFs with 4 ORFs in virion-sense DNA and 4 ORFs in complementary-sense DNA; Y8 DNAβ consists of 1338 nucleotides encoding 7 predicted ORFs with 4 ORFs in virion-sense DNA and 3 ORFs in complementary-sense DNA. TLCV DNAβ has little sequence homology to DNA-A of TLCV., except that it shares conserved TAATATTAC loop sequence with TLCV DNA-A. Sequence comparison showed that Y5 DNAβ shared 85% sequence homology with Y8 DNAβ, and both Y5 DNAβ and Y8 DNAβ had relatively low sequence identity (51%–65%) with the reported DNAβ molecules associated with Ageratum yellow vein virus and Cotton leaf curl virus. The immunotrapping PCR and whitefly transmission tests showed that DNAβ molecule could be encapsidated in virus particle and transmitted by Bemisia tabaci. This is the first report of DNAβ associated with WTGs in China.