Cotton Leaf Curl Gezira Betasatellite Research Articles

Demonstration of Insect Vector-Mediated Transfer of a Betasatellite between Two Helper Viruses.

Begomoviruses, transmitted by the whitefly Bemisia tabaci, pose significant threats to global agriculture due to their severe impact on various crops. Among the satellite molecules associated with begomoviruses, betasatellites play a crucial role in enhancing disease severity and yield losses. The spread and association of these molecules with helper viruses in host plants are thus matters of concern. Here, we focus on the propagation of betasatellites and, more specifically, on their transfer between different helper viruses and hosts through vector transmission. Our results show that the cotton leaf curl Gezira betasatellite (CLCuGeB), initially acquired with its helper virus cotton leaf curl Gezira virus (CLCuGeV) from an okra plant, can be transmitted and assisted by a different helper virus, tomato yellow leaf curl virus (TYLCV), in a different host plant (tomato plant). The new association can be formed whether TYLCV and CLCuGeB encounter each other in a host plant previously infected with TYLCV or in whiteflies having acquired the different components separately. Our findings reveal two pathways by which betasatellites can be transferred between helper viruses and host plants and highlight the ability of betasatellites to spread in begomovirus-infected environments.

First Report of the Satellite DNA Beta Associated with Tomato Yellow Leaf Curl Virus – Mild on Tomato in Iraq

In November 2021, severe symptoms of TYLCV disease were observed in tomato fields in Karbala Province, Iraq. Symptomatic tomato samples were observed on 20 to 30% of plants. Samples of symptomatic plants were collected randomly. TYLCV-specific PCR primers were employed to confirm the presence of the virus. Total DNA was extracted and used as templates for next-generation sequencing. The unmapped reads were subjected to de novo assembly. The contigs assembled were compared with GenBank data of plant viruses. A BLASTn analysis revealed 99.34% similarity with tomato yellow leaf curl virus-mild (MT583814.1). This finding was confirmed by a PCR test using specific primers. Additionally, there was 94.66% pairwise identity with cotton leaf curl Gezira betasatellite (CLCuGB) isolate Al-Ain (KM279620.1). The universal primer pair for detection of betasatellites was applied, followed by Sanger sequencing of the amplicons, which confirmed the infection of all the tested plants with CLCuGB. The DNA sequence of CLCuGB has accession number ON206651. To verify the presence of TYLCV-mild and betasatellite, a field survey was conducted in the main regions of tomato cultivation in Karbala Province, Iraq, in November 2021. Symptomatic leaf samples were collected randomly from 40 fields and analyzed for virus infections. This analysis revealed that the virus and its satellite are distributed throughout the province. To our knowledge, this is the first record of occurrence and complete genome sequence of CLCuGB, associated with TYLCV-mild, in tomato fields in Iraq.

Identification and molecular characterization of cotton leaf curl Gezira betasatellite and two distinct begomoviruses infecting papaya trees in the Kingdom of Saudi Arabia

Begomovirus is the largest genus in the family Geminiviridae and constitutes more than 445 virus species. Begomoviruses are characterized by single-stranded circular genomes with monopartite or bipartite components and transmitted by whitefly (Bemisia tabaci). Begomoviruses cause severe diseases in many economically important crops throughout the world. Typical symptoms of a begomovirus infection including severe leaf curling, vein thickening, vein darkening and reduced leaf size were observed in papaya plants in the Dammam district of the Eastern Province of Saudi Arabia during the growing season in 2022. A total of 10 samples were collected, and total genomic DNA was isolated from naturally infected papaya tree samples and subjected to PCR amplification using universal diagnostic primers for begomoviruses and associated satellites. Three PCR-amplified genomic components of begomoviruses and betasatellite namely P61Begomo (645 bp), P62Begomo (341 bp) and P62Beta (563 bp) were sent for Sanger DNA sequencing to Macrogen Inc. These partial viral genome sequences were submitted to Genbank database and accession numbers ON206051, ON206052 and ON206050 were assigned to P61Begomo, P62Begomo and P62Beta respectively. Phylogenetic analysis and pairwise nucleotide sequence identity studies identified P61Begomo was identified as Tomato yellow leaf curl virus, P62Begomo as DNA A component of a bipartite begomovirus Watermelon chlorotic stunt virus and P62Beta as begomovirus associated betasatellite; Cotton leaf curl Gezira betasatellite. To the best of our knowledge, this is the first report of a begomovirus complex infecting papaya (Carica papaya) in the Kingdom of Saudi Arabia.

Complete nucleotide sequence ofa 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.

First Report of Cotton leaf curl Gezira virus and Its Associated Alphasatellite and Betasatellite from Disease Affected Okra Plants in the United States

Okra (Abelmoschus esculentus [L.] Moench) is an important fresh market vegetable crop in southern Texas, U.S.A. In fall 2018, an investigation was conducted to identify the agent(s) of a virus-like disease outbreak on a 4.9-ha okra (cv. Clemson Spineless) farm in Hidalgo Co., Texas. By mid-growing season, disease incidence was 100%, which led to total crop loss. Symptoms—foliar yellow mosaic, bleaching, and vein clearing—were reminiscent of those caused by begomoviruses (Begomovirus, Geminiviridae). Leaves collected from 10 randomly distributed symptomatic plants were subjected to total DNA isolation (Dellaporta et al. 1983) and rolling cycle amplification (RCA) (TempliPhi 100 Amplification Kit, GE Healthcare Biosciences). The RCA product was used as a template for PCR amplification of a diagnostic fragment of the DNA-A (Wyatt and Brown 1996) and DNA-B (Rojas et al. 1993) components and full-length alphasatellites (Bull et al. 2003) and betasatellites (Briddon et al. 2002). The expected-size amplicon corresponding to each of the four targets was obtained from all 10 samples, except the betasatellite, for which half of the expected ∼1,350-bp product resulted. Amplicons were cloned into the pJET1.2 plasmid vector, and inserts of the expected sizes were Sanger sequenced (n ≥ 3 per event). BLASTn searches of GenBank revealed an unexpectedly complex begomovirome, consisting of the monopartite cotton leaf curl Gezira virus (CLCuGV), cotton leaf curl Gezira alphasatellite (CLCuGA; Colecusatellite, Alphasatellitidae), cotton leaf curl Gezira betasatellite (CLCuGB; Betasatellite, Tolecusatellitidae), and the bipartite okra yellow mosaic Mexico virus (OkYMMV). Viral genome-, component-, and satellite-specific abutting primers were designed and used to PCR amplify the corresponding molecule from two RCA products in two different labs, followed by cloning and bidirectional Sanger sequencing of ≥3 clones per event. The complete genome sequences were analyzed comparatively with corresponding GenBank sequences of each genomic DNA segment using SDT1.2 (Muhire et al. 2014). The OkYMMV DNA-A of 2,613 nt (GenBank MN027195 to 96) and DNA-B of 2,594 nt (MN027197 to 98), each shared 99.5 and 99.7% maximum identity, respectively, with the DNA-A (HM035059) and -B (HM035060) of OkYMMV-[US:TX:09] from okra plants in Texas (Hernandez-Zepeda et al. 2010). The complete monopartite genome sequence of the CLCuGV okra isolates (MN027199 to 202) was 2,764 nt and shared 99.5 to 99.6% maximum identity with an isolate of CLCuGV from hollyhock plants in Jordan (CLCuGV-[JO:Hol:09]: GU945265). The closest relative of the CLCuGA okra isolate, of 1,354 nt (MN027203 to 05), was CLCuGA-[IL:IsSq-C171:11] (KT099176) from whiteflies collected from squash plants in Israel, at 99% identity. Several approaches by two independent laboratories to clone and sequence a full-length CLCuGB, involving restriction digestion from total DNA or RCA (n = 2) and/or PCR amplification with abutting primers designed based on the full-length CLCuGB sequences (AF397217, KT099178), yielded only “defective-sized” CLCuGB molecules of 676 to 730 nt (MN027206 to 220). The betasatellite half-mers lacked the βC1 coding region while sharing 96 to 99% identity with CLCuGB-[EG:OK03:02] (AF397217) from okra plants in Egypt and CLCuGB-[IL:IsSq-C9:11] (KT099178) from whiteflies. This is the first U.S. report of CLCuGV and its associated alphasatellite and betasatellite, which are endemic to Africa (Idris and Brown 2000). More recently, members of the CLCuGV-satellite complex have been reported in the African Sahel, Arabian Peninsula and Middle East, and Pakistan (Idris et al. 2014; Tahir et al. 2011; Tiendrebeogo et al. 2010). The source of this Old World CLCuGV-satellite complex into New World okra plants is unknown, but its introduction via infected plants or viruliferous whiteflies transported into southern Texas, with its intensive cotton and vegetable production, is plausible. Spread of this complex poses a serious threat because U.S. cotton and vegetable varieties harbor no resistance to CLCuGV.

The recent association of a DNA betasatellite with Tomato yellow leaf curl virus in Israel – A new threat to tomato production

During November 2016, tomato yellow leaf curl virus (TYLCV)-resistant F1 hybrid indeterminate tomato plants cv. Diagrama, grown in a commercial greenhouse in Revaya, Israel, expressed severe symptoms of tomato yellow leaf curl disease (TYLCD). Samples were collected from symptomatic tomato plants, and using TYLCV-specific PCR primers, it was confirmed that these plants were indeed infected with TYLCV. Using the universal betasatellite primer pair B01/B02, followed by cloning and sequencing, it was found that the plants were also infected with a betasatellite. The satellite sequence was deposited in NCBI (GeneBank Acc. No. MK456609). Phylogenetic analysis revealed that the closest similarity, with 96% nucleotide identity, was to cotton leaf curl Gezira betasatellite (CLCuGB). Sampling symptomatic tomato plants in major growing areas in Israel revealed that CLCuGB is disseminated throughout the country. To the best of our knowledge, this is the first identification of a betasatellite, associated with TYLCV, in tomato plants in Israel.

Compatibility and interaction of begomoviruses and DNA-satellites causing leaf curl disease in Asia, Africa and Mediterranean Region

A complex of begomoviruses and their associated DNA-satellites is causing leaf curl diseases in Asia, Africa and the Mediterranean Region. Begomoviruses and DNA-satellites native to Asia and Africa are distinct from each other. Different combinations of geographically distinct monopartite begomoviruses, betasatellites and an alphasatellite were analyzed in infection experiments: cotton leaf curl Kokhran virus (CLCuKoV) and cotton leaf curl Multan virus (CLCuMuV) from Pakistan along with their associated cotton leaf curl Multan betasatellite (CLCuMuB); okra yellow crinkle virus (OYCrV), cotton leaf curl Gezira betasatellite (CLCuGeB), ageratum leaf curl Cameroon betasatellite (ALCCMB) and ageratum leaf curl Cameroon alphasatellite (ALCCMA) from Cameroon; tomato yellow leaf curl virus (TYLCV) from the Mediterranean Region. The infectivity assays were carried out in the model host species Nicotiana benthamiana and agro-infectious clones containing partial tandem repeats of the begomovirus and satellite genomes. The tested begomoviruses from different regions could functionally interact with non-cognate betasatellites and alphasatellite. Plants co-inoculated with CLCuKoV/CLCuGeB or CLCuMuV/CLCuGeB developed, for both combinations, more severe symptoms than with the begomovirus alone and the infection was verified by Southern blot hybridization. Nonetheless, CLCuKoV and CLCuMuV were shown also to support infection by ALCCMB, whereas OYCrV and TYLCV were able to support infection with CLCuMuB, ALCCMB and CLCuGeB. Moreover, all begomoviruses from different geographical origins supported ALCCMA from Africa. The results confirm that geographically diverse begomoviruses can functionally interact with non-cognate DNA-satellites, which may cause the emergence of new begomovirus-satellite complexes. Possibly, exchange of DNA-satellites may occur across continents leading to the emergence of new viral diseases.

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.

Charlock mustard (Sinapis arvensis): a weed reservoir for begomoviruses and associated betasatellite in Jordan

Four crop-infecting begomoviruses were found naturally affecting Charlock mustard (Sinapis arvensis L.) plants in Jordan and characterized at the molecular level. Symptoms included leaf curling and stunting. PCR analysis showed the presence of Tomato yellow leaf curl virus (TYLCV), Tomato yellow leaf curl Sardinia virus (TYLCSV), Squash leaf curl virus (SLCV) and Watermelon chlorotic stunt virus (WmCSV). These viruses were associated with Cotton leaf curl Gezira betasatellite (CLCuGβ). Sequence analysis revealed that TYLCV and TYLCSV had 98.5% and 99.3% nucleotide (nt) sequence identities with a previously described TYLCV strain from Florida and TYLCSV from Sardinia, respectively. The other two viruses, SLCV and WmCSV, shared 99.7% and 99.5% nt identities with previously described strains from Jordan. Sequence analysis also showed that CLCuGβ had 98.8% nt identity with a strain from Israel. To our knowledge, this is the first report of S. arvensis as a natural host of SLCV, WmCSV, TYLCSV and CLCuGβ.

Begomovirus disease complex: emerging threat to vegetable production systems of West and Central Africa

Vegetables play a major role in the livelihoods of the rural poor in Africa. Among major constraints to vegetable production worldwide are diseases caused by a group of viruses belonging to the genus Begomovirus, family Geminiviridae. Begomoviruses are plant-infecting viruses, which are transmitted by the whitefly vector Bemisia tabaci and have been known to cause extreme yield reduction in a number of economically important vegetables around the world. Several begomoviruses have been detected infecting vegetable crops in West and Central Africa (WCA). Small single stranded circular molecules, alphasatellites and betasatellites, which are about half the size of their helper begomovirus genome, have also been detected in plants infected by begomoviruses. In WCA, B. tabaci has been associated with suspected begomovirus infections in many vegetable crops and weed species. Sequencing of viral genomes from crops such as okra resulted in the identification of two previously known begomovirus species (Cotton leaf curl Gezira virus and Okra yellow crinkle virus) as well as a new recombinant begomovirus species (Okra leaf curl Cameroon virus), a betasatellite (Cotton leaf curl Gezira betasatellite) and new alphasatellites. Tomato and pepper plants with leaf curling were shown to contain isolates of new begomoviruses, collectively referred to as West African tomato-infecting begomoviruses (WATIBs), new alphasatellites and betasatellites. To study the potential of weeds serving as begomovirus reservoirs, begomoviruses and satellites in the weed Ageratum conyzoides were characterized. Sequence analyses showed that they were infected by isolates of a new begomovirus (Ageratum leaf curl Cameroon virus) that belong to the WATIBs group, a new betasatellite (Ageratum leaf curl Cameroon betasatellite), an alphasatellite and two types of defective recombinants between a begomovirus and an alphasatellite. Putative recombinations were detected in begomovirus genomes for all four plant species studied, indicating that recombination is an important mechanism for their evolution. A close relationship between the begomoviruses infecting pepper and tomato and A. conyzoides and the detection of the same alphasatellite in them support the idea that weeds are important reservoirs for begomoviruses and their satellites. With this high diversity, recombination potential and transmission by B. tabaci, begomoviruses and ssDNA satellites pose a serious threat to crop production in West and Central Africa.

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.

Ageratum conyzoides: A host to a unique begomovirus disease complex in Cameroon

Ageratum conyzoides (goat weed) is a widespread uncultivated species in Cameroon that exhibits leaf curl disease (LCD) symptoms suggestive of begomovirus infection. In Asia, different begomovirus–satellite complexes have been identified in A. conyzoides. The objective of this study was to determine the identity of the suspect begomoviruses and their associated satellites in A. conyzoides in Cameroon. The results indicated that all three symptomatic A. conyzoides plants examined were infected with a new begomovirus species, herein named Ageratum leaf curl Cameroon virus (ALCCMV). The ALCCMV genome sequences shared their highest identity, at 84.3–88.5%, with a group of tomato-infecting begomoviruses from West Africa. In addition, a betasatellite and an alphasatellite were cloned from the same symptomatic A. conyzoides plants. The betasatellite sequences shared limited sequence identity at 37% or less with the betasatellite Cotton leaf curl Gezira betasatellite, and the new betasatellite species is herein named Ageratum leaf curl Cameroon betasatellite (ALCCMB). The alphasatellite shared 80% nt identity with Tomato leaf curl Cameroon alphasatellite (ToLCCMA), and the new alphasatellite species is herein named Ageratum leaf curl Cameroon alphasatellite (ALCCMA). In addition, two fragments containing begomovirus–alphasatellite sequences were cloned from sample AGLI4, and they were related to the defecting interfering molecule (Y14167) associated with Ageratum yellow vein virus from Asia. These results suggest that the begomoviral–satellite complexes infecting A. conyzoides in Cameroon may be as complex or more so, to species and strains reported thus far from Asia.

The merging of two dynasties--identification of an African cotton leaf curl disease-associated begomovirus with cotton in Pakistan.

Cotton leaf curl disease (CLCuD) is a severe disease of cotton that occurs in Africa and Pakistan/northwestern India. The disease is caused by begomoviruses in association with specific betasatellites that differ between Africa and Asia. During survey of symptomatic cotton in Sindh (southern Pakistan) Cotton leaf curl Gezira virus (CLCuGV), the begomovirus associated with CLCuD in Africa, was identified. However, the cognate African betasatellite (Cotton leaf curl Gezira betasatellite) was not found. Instead, two Asian betasatellites, the CLCuD-associated Cotton leaf curl Multan betasatellite (CLCuMB) and Chilli leaf curl betasatellite (ChLCB) were identified. Inoculation of the experimental plant species Nicotiana benthamiana showed that CLCuGV was competent to maintain both CLCuMB and ChLCB. Interestingly, the enations typical of CLCuD were only induced by CLCuGV in the presence of CLCuMB. Also in infections involving both CLCuMB and ChLCB the enations typical of CLCuMB were less evident. This is the first time an African begomovirus has been identified on the Indian sub-continent, highlight the growing threat of begomoviruses and particularly the threat of CLCuD causing viruses to cotton cultivation in the rest of the world.

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.

A severe symptom phenotype in tomato in Mali is caused by a reassortant between a novel recombinant begomovirus (Tomato yellow leaf curl Mali virus) and a betasatellite

Tomato production in West Africa has been severely affected by begomovirus diseases, including yellow leaf curl and a severe symptom phenotype, characterized by extremely stunted and distorted growth and small deformed leaves. Here, a novel recombinant begomovirus from Mali, Tomato yellow leaf curl Mali virus (TYLCMLV), is described that, alone, causes tomato yellow leaf curl disease or, in combination with a betasatellite, causes the severe symptom phenotype. TYLCMLV is an Old World monopartite begomovirus with a hybrid genome composed of sequences from Tomato yellow leaf curl virus-Mild (TYLCV-Mld) and Hollyhock leaf crumple virus (HoLCrV). A TYLCMLV infectious clone induced leaf curl and yellowing in tomato, leaf curl, crumpling and yellowing in Nicotiana benthamiana and common bean, mild symptoms in N. glutinosa, and a symptomless infection in Datura stramonium. In a field-collected sample from a tomato plant showing the severe symptom phenotype in Mali, TYLCMLV was detected together with a betasatellite, identified as Cotton leaf curl Gezira betasatellite (CLCuGB). Tomato plants co-agroinoculated with TYLCMLV and CLCuGB developed severely stunted and distorted growth and small crumpled leaves. These symptoms were more severe than those induced by TYLCMLV alone, and were similar to the severe symptom phenotype observed in the field in Mali and in other West African countries. TYLCMLV and CLCuGB also induced more severe symptoms than TYLCMLV in the other solanaceous hosts, but not in common bean. The increased symptom severity was associated with hyperplasia of phloem-associated cells, but relatively little increase in TYLCMLV DNA levels. In surveys of tomato virus diseases in West Africa, TYLCMLV was commonly detected in plants with leaf curl and yellow leaf curl symptoms, whereas CLCuGB was infrequently detected and always in association with the severe symptom phenotype. Together, these results indicate that TYLCMLV causes tomato yellow leaf curl disease throughout West Africa, whereas TYLCMLV and CLCuGB represent a reassortant that causes the severe symptom phenotype in tomato.

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-β 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.