Complete nucleotide sequence of a begomovirus associated with an alphasatellite and a betasatellite naturally infecting okra in Jordan.

The complete nucleotide sequences of a monopartite begomovirus and an associated alphasatellite and betasatellite isolated from naturally infected okra (Abelmoschus esculentus) plants originating from Jordan were determined. The sequences of the begomovirus, alphasatellite, and betasatellites were determined to be 2,764, 1,307, and 1,354 nucleotides in length, respectively. Sequence Demarcation Tool (SDT) and phylogenetic analysis revealed that the begomovirus isolate shared the highest (99.5-99.8%) nt sequence identity with isolates of cotton leaf curl Gezira virus (CLCuGeV), a begomovirus found to exclusively infect cotton in Africa, and recently, in Asia and the Middle East. The DNA sequences of the alphasatellite and betasatellite exhibited the highest nt sequence identity (98.7-98.9% and 92.2-95.3%, respectively) to cotton leaf curl Gezira alphasatellite and cotton leaf curl Gezira betasatellite, respectively. This is the first identification of an African begomovirus, associated with DNA satellites, infecting okra in Jordan.

Two different begomovirus species are associated with yellow vein mosaic disease of okra in Sri Lanka.

Yellow vein mosaic disease is the major biotic constraint of okra cultivation in Sri Lanka. Identification and detailed molecular characterization of associated pathogen is needed for effective disease management. The genome of the begomovirus and betasatellite were amplified in symptomatic plant samples using specific degenerate primers. DNA-A genome of twelve isolates representing different locations in Sri Lanka were cloned, sequenced and deposited in GenBank database (Accession No- KX698087- KX698092 and MH455207- MH455212). Size of the complete nucleotide sequences ranged from 2735 to 2786 bp. The genome organization showed characteristics of begomoviruses. The pairwise sequence identity revealed the association of two different begomovirus species. Five of the isolates showed > 91% of sequences identity with Bhendi yellow vein mosaic virus, and the rest of the seven isolates were around 92% of identity with Okra enation leaf curl virus. This is further supported by phylogenetic analysis where both of these group of isolates were in different cluster. Recombination analysis showed the presence of recombinant fragments in the virus isolates associated with okra yellow vein mosaic disease (OYVMD) in Sri Lanka. Attempts to amplify DNA- B were failed in any of the samples tested. However, both type of the begomovirus species associated with betasatellite species, Bhendi yellow vein mosaic betasatellite. The present study has revealed the association of two distinct monopartite begomovirus species, Bhendi yellow vein mosaic virus or Okra enation leaf curl virus, with OYVMD in Sri Lanka.

Inheritance pattern of okra enation leaf curl disease among cultivated species and its relationship with biochemical parameters.

Okra production in eastern India at present is severely threatened by whitefly-mediated okra enation leaf curl disease (OELCuD). Identification of resistant genotype and understanding the genetic control and biochemical relationship of OELCuD resistance are prerequisite for developing an effective breeding strategy. This study was conducted employing six populations (P1, P2, F1, F2, BC1 and BC2) of two selected (resistant x susceptible (RxS)) crosses. Associationship between severity of OELCuD and biochemical parameters of parents and hybrids at preflowering and flowering stages was studied. Segregation pattern of the genotypes in F2 generation showing OELCuD reaction of two crosses suggested that two duplicate recessive genes was operative for resistance to OELCuD. Generation mean analysis revealed involvement of both additive and nonadditive effects in the inheritance of disease resistance. Hence, postponement of selection in later generations or intermating among the selected segregates followed by one or two generations of selfing to break the undesirable linkage and allow the accumulation of favourable alleles could be suggested for the development of stable resistant genotype against this disease. Higher peroxidase activity and total phenol content in leaf emerged as reliable biochemical markers for early selection of genotype resistant to OELCuD.

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.

Detection and absolute quantification of betasatellites associated with okra yellow vein mosaic disease by qPCR.

Okra yellow vein mosaic disease (OYVMD) causes serious loss in okra production in Sri Lanka. Therefore, screening of resistant okra verities is an essential need to control the disease. As the available qualitative and semi-quantitative methods failed to detect latent infection the present study aimed to develop a quantitative PCR (qPCR) assay to detect and quantify one of the OYVMD causing agent, symptom modulating satellite molecules. A pair of primers targeting a portion of ßC1 gene of BYVMBs was designed and used to quantify of BYVMBs by absolute quantification method using SYBR Green I chemistry. Standard curves were prepared using series of dilutions of known copy number plasmids carrying target sequence. The mean amplification efficiency was 95% and the coefficient of determination was 0.994. The method was tested to find out the relation between symptoms and betasatellite titre in range of severity of OYVMD symptoms; the betasatellite titre increased with increasing severity. Interestingly, the method was able to detect BYVMBs present in apparently healthy plants growing in an infected field at a concentration which was not able to detect in end point PCR. Betasatellite titre was also measured in different ages of leaves and different positions. On average, the betasatellite titre in younger leaves was higher than in mature leaves and there were no significant variations in betasatellite titre in different position in each leaf. The assay was also tested as a tool to screen for resistant okra varieties; among the eight varieties tested no BYVMBs were detected in variety Maha F1. Varieties TV8 and MI5 had significantly higher copy number than rest of the varieties. The qPCR protocol described in this study is a useful method to detect and quantify BYVMBs in okra, especially for plant samples with betasatellite titre lower than the detection limit of conventional methods.

Infectivity of okra enation leaf curl virus and the role of its V2 protein in pathogenicity.

Cotton crop has been severely affected by multiple begomoviruses in Pakistan and India. In our previous study, we found okra enation leaf curl virus (OELCuV), cotton leaf curl Multan betasatellite (CLCuMuB) and cotton leaf curl Multan alphasatellite (CLCuMuA) infecting cotton in Pakistan. The current study was designed to investigate the infectivity of OELCuV and its ability to trans-replicate non-cognate CLCuMuB. Agro-infectious clones containing the partial tandem repeats of OELCuV and CLCuMuB were constructed and the infectivity assays were carried out through Agrobacterium mediated transformation in the model host species Nicotiana benthamiana under controlled conditions. The results showed that in the inoculated plants OELCuV alone can cause downward curling and yellowing of leaves with thickened veins. However, when co-inoculated with the non-cognate CLCuMuB it could functionally trans-replicate CLCuMuB resulting in a more severe phenotype. The expression of Pre-coat/V2 protein in the N. benthamiana plants through the potato virus X (PVX) system caused localized cell death after severe leaf curling in the infiltrated leaves. The tissue tropism of the virus was associated with the systemic development of a hypersensitive response (HR), which ultimately lead to the plant death. The results indicated the involvement of V2 protein in the pathogenicity of OELCuV and its ability to trigger the host defense machinery. This study also demonstrated the ability of OELCuV to trans-replicate CLCuMuB resulting in typical leaf curl disease symptoms in N. benthamiana.

Accumulation and transmission of alphasatellite, betasatellite and tomato yellow leaf curl virus in susceptible and Ty-1-resistant tomato plants.

Begomoviruses (family Geminiviridae) are frequently associated with alphasatellites and betasatellites in the Old World. Tomato yellow leaf curl virus, one of the most damaging begomovirus species worldwide, was recently found associated with betasatellites in the eastern coast of the Mediterranean Sea, and in the Middle East region. Tomato yellow leaf curl virus (TYLCV)/betasatellite associations were shown to increase TYLCV virulence in experimental conditions. The sustainability of TYLCV/satellite associations in tomato was assessed here by estimating accumulation levels of satellites in comparison to TYLCV, vector transmission efficiency, and by testing how far the popular Ty-1 resistance gene used in most TYLCV-resistant tomato cultivars in the Mediterranean Basin is effective against betasatellites. Three satellites previously isolated from okra in Burkina Faso-of the species Cotton leaf curl Gezira betasatellite, Cotton leaf curl Gezira alphasatellite and Okra leaf curl Burkina Faso alphasatellite-were shown to accumulate at levels similar to, or higher than, the helper virus TYLCV-Mld in tomato plants from 32 to 150 days post inoculation (dpi). Cotton leaf curl Gezira betasatellite (CLCuGB) reduced TYLCV-Mld accumulation whereas alphasatellites did not. Transmission tests were performed with B. tabaci from plants infected with TYLCV-Mld/CLCuGB- or TYLCV-Mld/Okra leaf curl Burkina Faso alphasatellite. At 32 dpi, both satellites were transmitted to more than 50% of TYLCV-infected test plants. Betasatellite transmission, tested further with 150 dpi source plants was successful in more than 30% of TYLCV-infected test plants. Ty-1 resistant tomato plants co-infected with TYLCV (-Mld or -IL) and CLCuGB exhibited mild leaf curling and mosaic symptoms at the early stage of infection associated with a positive effect on TYLCV-IL accumulation, while resistant plants infected with TYLCV only, were asymptomatic. Together with previous experimental studies, these results further emphasize the potential risk of betasatellites to tomato cultivation, including with Ty-1 resistant cultivars.

Genetic control of yellow vein mosaic virus disease tolerance in Abelmoschus esculentus (L.) Moench.

Okra's (Abelmoschus esculentus (L.) Moench) commercial cultivation is threatened in the tropics due to high incidence of yellow vein mosaic virus (YVMV) disease. Okra geneticists across the world tried to understand the inheritance pattern of YVMV disease tolerance without much success. Therefore, the inheritance pattern of YVMV disease in okra was revisited by employing sixgenerations (P1, P2, F1, F2, BC1 and BC2) of four selected crosses (one tolerant × tolerant, two tolerant × susceptible and one susceptible × susceptible) using two tolerant (BCO-1 and Lal Bhendi) and two susceptible (Japanese Jhar Bhendi and PAN 2127) genotypes. Qualitative genetic analysis was done on the basis of segregation pattern of tolerant and susceptible plants in F2 and backcross generations of all the four crosses. It revealed that a single dominant gene along with some minor factors governed the disease tolerant trait in both the tolerant parents used. However, it was observed that genes governing disease tolerance identified in both the tolerant variety used was different. It could be concluded that the gene governing YVMV disease tolerance in okra wasgenotype specific. Further, duplicate gene action as evident from an approximate ratio of 15:1 (tolerant:susceptible) in the F2 population in the cross of two tolerant varieties gave a scope of increasing the tolerance level of the hybrid plants when both the tolerant genes are brought together. However, generation mean analysis revealed involvement of both additive and nonadditive effects in the inheritance of disease tolerance. Thus, the present study confirms that a complicated genetic inheritance pattern is involved in the disease tolerance against YVMV trait. The major tolerance genes could be transferred to other okra varieties, but the tolerance breaking virus strains might not allow them to achieve tolerance in stable condition. Therefore, accumulation of additional genes may be needed for a sustainable tolerance phenotype in okra.

Molecular diversity, recombination and population structure of alphasatellites associated with begomovirus disease complexes.

The genus, begomovirus (family Geminiviridae) includes a large number of viruses infecting a wide range of plant species worldwide. The majority of monopartite begomoviruses are associated with satellites (betasatellites) and/or satellite-like molecules (alphasatellites). In spite of the Indo-China region being regarded as the centre of origin of begomoviruses and satellites, a detailed study on the emergence and evolution of alphasatellites in India has not yet conducted. Our present analysis indicated the association of 22 alphasatellites with monopartite and bipartite begomovirus-betasatellite complexes in India. Based on sequence pairwise identity, these alphasatellites were categorized into five distinct groups: Cotton leaf curl alphasatellite, Gossypium darwinii symptomless alphasatellite, Gossypium mustelinum symptomless alphasatellite, Okra leaf curl alphasatellite and an unreported Chilli leaf curl alphasatellite (ChiLCA). Furthermore, infectivity analysis of the cloned ChiLCA along with the viral components of either cognate or non-cognate chilli-infecting begomoviruses on Nicotiana benthamiana suggested that ChiLCA is dispensable for leaf curl disease development. It is noteworthy that in the presence of ChiLCA, a marginal decrease in betasatellite DNA level was noticed. Additionally, high genetic variability and diverse recombination patterns were detected among these alphasatellites, and the nucleotide substitution rate for the Rep gene of ChiLCA was determined to be 2.25×10-3nucleotides/site/year. This study highlights the genetic distribution, and likely contribution of recombination and nucleotide diversity in facilitating the emergence of alphasatellites.

Molecular genetic analysis and evolution of begomoviruses and betasatellites causing yellow mosaic disease of bhendi.

In India, Bhendi yellow vein mosaic disease (BYVMD) is one of the most economically important diseases of bhendi/okra and is caused by a complex of monopartite begomovirus (Bhendi yellow vein mosaic virus-BYVMV) and betasatellite (Bhendi yellow vein betasatellite-BYVB). In this study, we have analyzed the role of possible evolutionary factors involved in the evolution of BYVMV and BYVB isolates. Evidence of inter-species and inter-strain recombination events was detected among the viral isolates, and majority of these recombinant isolates possess microsatellites in their genome. Recombination analysis suggests that cotton-infecting and bhendi-infecting begomoviruses probably share a recent common ancestor. In addition to genetic differentiation and gene flow, high degree of genetic variability was detected among the viral population. A strong purifying selection seems to be acting on the viral coding regions. The nucleotide substitution rate of V1 gene (for BYVMV) and ßC1 gene (for BYVB) was estimated to be 7.55 × 10-4 and 2.25 × 10-3 nucleotide substitutions/site/year, respectively. The present study underlines that the evolution of BYVMD-associated viral components is driven by selection acting on the genetic variation generated by recombination and mutation.

Association of tomato leaf curl New Delhi virus DNA-B with bhendi yellow vein mosaic virus in okra showing yellow vein mosaic disease symptoms.

Okra samples showing yellow vein mosaic, vein twisting and bushy appearance were collected from different locations of India during the surveys conducted between years 2005-2009. The dot blot and PCR detection revealed that 75.14% of the samples were associated with monopartite begomovirus and remaining samples with bipartite virus. Whitefly transmission was established for three samples representing widely separated geographical locations which are negative to betasatellites and associated with DNA-B. Genome components of these three representative isolates were cloned and sequenced. The analysis of DNA-A-like sequence revealed that three begomovirus isolates shared more than 93% nucleotide sequence identity with bhendi yellow vein mosaic virus from India (BYVMV), a monopartite begomovirus species that was reported previously as causative agent of bhendi yellow mosaic disease in association of bhendi yellow vein mosaic betasatellite. Further, the DNA-B-like sequences associated with the three virus isolates shared no more than 90% sequence identity with tomato leaf curl New Delhi virus (ToLCNDV). Analyses of putative iteron-binding sequence required for trans-replication suggests that begomovirus sequences shared compatible rep-binding iterons with DNA-B of ToLCNDV. Our data suggest that the monopartite begomovirus associated with okra yellow vein disease has captured DNA-B of ToLCNDV to infect okra. Widespread distribution of the complex shows the increasing trend of the capturing of DNA-B of ToLCNDV by monopartite begomoviruses in the Indian subcontinent. The recombination analysis showed that the DNA-A might have been derived from the inter-specific recombination of begomoviruses, while DNA-B was derived from the ToLCNDV infecting different hosts.

Identification of a disease complex involving a novel monopartite begomovirus with beta- and alphasatellites associated with okra leaf curl disease in Oman.

Okra leaf curl disease (OLCD) is an important viral disease of okra in tropical and subtropical areas. The disease is caused by begomovirus-satellite complexes. A begomovirus and associated betasatellite and alphasatellite were identified in symptomatic okra plants from Barka, in the Al-Batinah region of Oman. Analysis of the begomovirus sequences showed them to represent a new begomovirus most closely related to cotton leaf curl Gezira virus (CLCuGeV), a begomovirus of African origin. The sequences showed less than 85 % nucleotide sequence identity to CLCuGeV isolates. The name okra leaf curl Oman virus (OLCOMV) is proposed for the new virus. Further analysis revealed that the OLCOMV is a recombinant begomovirus that evolved by the recombination of CLCuGeV isolates with tomato yellow leaf curl virus-Oman (TYLCV-OM). An alpha- and a betasatellite were also identified from the same plant sample, which were also unique when compared to sequences available in the databases. However, although the betasatellite appeared to be of African origin, the alphasatellite was most closely related to alphasatellites originating from South Asia. This is the first report of a begomovirus-satellite complex infecting okra in Oman.

First report of an alphasatellite associated with Okra enation leaf curl virus.

An alphasatellite DNA associated with Okra enation leaf curl virus (OELCuV) which causes enation and leaf curling in okra (Abelmoschus esculentus) plants was characterized. The full-length DNA comprises 1,350 nucleotides and shows typical genome organization of an alphasatellite. It shows the highest nucleotide sequence identity (79.7 %) to Hollyhock yellow vein virus-associated symptomless alphasatellite (HoYVSLA). This is the first report of the association of an alphasatellite with OELCuV from India.

Molecular characterization of begomoviruses and DNA satellites associated with okra leaf curl disease in Cameroon.

Okra leaf curl disease (OLCD) is the most important viral disease of okra in West Africa. In this study, a complex of begomoviruses and associated DNA satellites were identified in symptomatic okra plants from southwestern Cameroon. Sequence analyses showed that two of the plants (Lik1 and Njo5) were infected with a begomovirus being a recombinant of cotton leaf curl Gezira virus (CLCuGeV) and okra yellow crinkle virus (OYCrV). The recombinant genome shared highest nucleotide identity with isolates of CLCuGeV at 87.8% and is therefore considered to be member of a new begomovirus species, Okra leaf curl Cameroon virus (OLCuCMV). One plant (Mue5) was infected by a begomovirus with 95.8% nucleotide identy to CLCuGeV, while in the plants Lik1, Mue1 and Njo5, a begomovirus was identified showing highest nucleotide identity at 93.7% with OYCrV. The nucleotide comparisons and phylogenetic analyses suggest that these isolates represent new Cameroonian strains of CLCuGeV and OYCrV (CLCuGeV-CM and OYCrV-CM). Mixed infection of OLCuCMV and OYCrV-CM was found in two of the plants. A betasatellite and two divergent alphasatellites were also associated with the begomoviruses. The betasatellite was identified as cotton leaf curl Gezira betasatellite (CLCuGeB) with the highest nucleotide identity at 93.3% to other African isolates of CLCuGeB. The alphasatellites, herein named Alpha-1 and Alpha-2, shared 97.3% and 95.2% identity, respectively, with cotton leaf curl Gezira alphasatellite (CLCuGeA) and okra leaf curl Burkina Faso alphasatellite (OLCuBFA). These collective results emphasize the extent of diversity among okra-infecting begomovirus-satellite complexes in western Africa.

Further evidence reveals that okra mottle virus arose from a double recombination event.

As a result of surveys of okra begomoviruses (genus Begomovirus, family Geminiviridae) conducted over the last five years in Central Brazil, we report the complete genome sequence of an isolate of okra mottle virus (OMoV). The DNA-A and DNA-B components were 2660 and 2653 nucleotides (nt) long, respectively, and they were most closely related to the DNA-A (~99 % nt identity) and DNA-B (~98 % nt identity) components of an OMoV isolate from a soybean plant. A phylogenetic tree was generated based on these sequences, and it was shown that both of the OMoV DNA components were grouped in a branch with Brazilian begomoviruses known to infect weeds. By recombination analysis, strong evidence was observed that the OMoV genome may have been the product of a double inter-species recombination event.

Molecular characterization of distinct bipartite begomovirus infecting bhendi (Abelmoschus esculentus L.) in India.

Yellow vein mosaic disease of okra is a whitefly transmitted begomovirus causing heavy economic loss in different parts of India. The okra isolate (OY131) of this virus from a bhendi plant [(Abelmoschus esculentus L.) Moench] showing yellow vein mosaic, vein twisting, reduced leaves, and a bushy appearance in the Palem region, New Delhi, India, was characterized in the present study. The complete DNA-A and DNA-B sequences have been determined and are comprised of 2,746 and 2,703 nucleotides, respectively. The betasatellite (DNA-ß) component was absent in the sample. The genome organization was typically of biparite begomoviruses, which were characterized earlier. Comparison of DNA-A component with other known begomoviruses suggest that this virus, being only distantly related (<85.9% similarity with its nearest relative, BYVMV) to other known begomoviruses, is a new species. We have tentatively assigned the genome to a novel geminivirus species Bhendi yellow vein mosaic Delhi virus [BYVDV-IN (India: Delhi: okra)]. DNA-B showed highest sequence identity (87.8% identical) to that of a ToLCNDV (AY158080). The phylogenetic analysis of the present isolate is distinct from all other viruses; however clusters with ToLCNDV group infect different crops. The recombination analysis revealed that this isolate has sequences originated from ToLCNDV. This is the first known bhendi yellow vein mosaic disease associated bipartite begomovirus from India.

Diversity and phylogeography of Begomovirus-associated beta satellites of Okra in India.

BACKGROUND: Okra (Abelmoschus esculentus; family Malvaceae) is grown in temperate as well as subtropical regions of the world, both for human consumption as a vegetable and for industrial uses. Okra yields are affected by the diseases caused by phyopathogenic viruses. India is the largest producer of okra and in this region a major biotic constraint to production are viruses of the genus Begomovirus. Begomoviruses affecting okra across the Old World are associated with specific, symptom modulating satellites (beta satellites). We describe a comprehensive analysis of the diversity of beta satellites associated with okra in India. RESULTS: The full-length sequences of 36 beta satellites, isolated from okra exhibiting typical begomovirus symptoms (leaf curl and yellow vein), were determined. The sequences segregated in to four groups. Two groups correspond to the beta satellites Okra leaf curl beta satellite (OLCuB) and Bhendi yellow vein beta satellite (BYVB) that have previously been identified in okra from the sub-continent. One sequence was distinct from all other, previously isolated beta satellites and represents a new species for which we propose the name Bhendi yellow vein India beta satellite (BYVIB). This new beta satellite was nevertheless closely related to BYVB and OLCuB. Most surprising was the identification of Croton yellow vein mosaic beta satellite (CroYVMB) in okra; a beta satellite not previously identified in a malvaceous plant species. The okra beta satellites were shown to have distinct geographic host ranges with BYVB occurring across India whereas OLCuB was only identified in northwestern India. Okra infections with CroYVMB were only identified across the northern and eastern central regions of India. A more detailed analysis of the sequences showed that OLCuB, BYVB and BYVIB share highest identity with respect ßC1 gene. ßC1 is the only gene encoded by beta satellites, the product of which is the major pathogenicity determinant of begomovirus-beta satellite complexes and is involved in overcoming host defenses based on RNAi. CONCLUSION: The diversity of beta satellites in okra across the sub-continent is higher than previously realized and is higher than for any other malvaceous plant species so far analyzed. The beta satellites identified in okra show geographic segregation, which has implications for the development and introduction of resistant okra varieties. However, the finding that the ßC1 gene of the major okra beta satellites (OLCuB, BYVB and BYVIB) share high sequence identity and provides a possible avenue to achieve a broad spectrum resistance.

Molecular diversity of cotton leaf curl Gezira virus isolates and their satellite DNAs associated with okra leaf curl disease in Burkina Faso.

Okra leaf curl disease (OLCD) is a major constraint on okra (Abelmoschus esculentus) production and is widespread in Africa. Using a large number of samples representative of the major growing regions in Burkina Faso (BF), we show that the disease is associated with a monopartite begomovirus and satellite DNA complexes. Twenty-three complete genomic sequences of Cotton leaf curl Gezira virus (CLCuGV) isolates associated with OLCD, sharing 95 to 99% nucleotide identity, were cloned and sequenced. Six betasatellite and four alphasatellite (DNA-1) molecules were also characterized. The six isolates of betasatellite associated with CLCuGV isolates correspond to Cotton leaf curl Gezira betasatellite (CLCuGB) (88 to 98% nucleotide identity). One isolate of alphasatellite is a variant of Cotton leaf curl Gezira alphasatellite (CLCuGA) (89% nucleotide identity), whereas the three others isolates appear to correspond to a new species of alphasatellite (CLCuGA most similar sequence present 52 to 60% nucleotide identity), provisionally named Okra leaf curl Burkina Faso alphasatellite (OLCBFA). Recombination analysis of the viruses demonstrated the interspecies recombinant origin of all CLCuGV isolates, with parents being close to Hollyhock leaf crumple virus (AY036009) and Tomato leaf curl Diana virus (AM701765). Combined with the presence of satellites DNA, these results highlight the complexity of begomoviruses associated with OLCD.

Roles and interactions of begomoviruses and satellite DNAs associated with okra leaf curl disease in Mali, West Africa.

Okra leaf curl disease (OLCD) is a major constraint on okra (Abelmoschus esculentus) production in West Africa. Two monopartite begomoviruses (okra virus-1 and okra virus-2), a betasatellite and a DNA1 satellite are associated with OLCD in Mali. Okra virus-1 is an isolate of okra yellow crinkle virus (OYCrV), okra virus-2 is a recombinant isolate of cotton leaf curl Gezira virus (CLCuGV) and the betasatellite is a variant of cotton leaf curl Gezira betasatellite (CLCuGB). Cloned DNA of OYCrV and CLCuGV were infectious and induced leaf curl symptoms in Nicotiana benthamiana plants, but did not induce OLCD in okra. However, when these clones were individually co-inoculated with the cloned CLCuGB DNA, symptom severity and viral DNA levels were increased in N. benthamiana plants and typical OLCD symptoms were induced in okra. The CLCuGB was also replicated by, and increased symptom severity of, three monopartite tomato-infecting begomoviruses, including two from West Africa. The sequence of the DNA1 satellite was highly divergent, indicating that it represents a distinct West African lineage. DNA1 replicated autonomously, and replication required the DNA1-encoded Rep protein. Although DNA1 reduced helper begomovirus DNA levels, symptoms were not attenuated. In the presence of CLCuGB, DNA levels of the helper begomoviruses and DNA1 were substantially increased. Together, these findings establish that OLCD in Mali is caused by a complex of monopartite begomoviruses and a promiscuous betasatellite with an associated parasitic DNA1 satellite. These findings are discussed in terms of the aetiology of OLCD and the evolution of new begomovirus/satellite DNA complexes.

Complete nucleotide sequences of okra isolates of Cotton leaf curl Gezira virus and their associated DNA-beta from Niger.

Okra (Abelmoschus esculentus) is a major crop in Niger. In the fall of 2007, okra leaf curl disease was observed in Niger and the begomovirus and DNA-beta satellite were found associated with the disease. The complete nucleotide sequences of DNA-A (FJ469626 and FJ469627) and associated DNA-beta satellites (FJ469628 and FJ469629) were determined from two samples. This is the first report of molecular characterization of okra-infecting begomovirus and their associated DNA-beta from Niger. The begomovirus and DNA-beta have been identified as Cotton leaf curl Gezira virus and Cotton leaf curl Gezira betasatellite, respectively, which are reported to also infect okra in Egypt, Mali and Sudan.