Cotton Leaf Curl Gezira Virus 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 cotton leaf curl Gezira virus infecting Malva parviflora and in Iraq

First report of cotton leaf curl Gezira virus infecting Malva parviflora and in Iraq

Incidence of cotton leaf curl Gezira virus and the associated alphasatellites and betasatellites in crops and ornamental plants in southern Iran

Incidence of cotton leaf curl Gezira virus and the associated alphasatellites and betasatellites in crops and ornamental plants in southern Iran

High-Throughput Sequencing Identified Distinct Bipartite and Monopartite Begomovirus Variants Associated with DNA-Satellites from Tomato and Muskmelon Plants in Saudi Arabia.

The studies on the prevalence and genetic diversity of begomoviruses in Saudi Arabia are minimal. In this study, field-grown symptomatic tomato and muskmelon plants were collected, and initially, begomovirus infection was confirmed by the core coat protein sequences. Four tomato and two muskmelon plants with viral infections were further evaluated for Illumina MiSeq sequencing, and twelve sequences (2.7-2.8 kb) equivalent to the full-length DNA-A or DNA-B components of begomoviruses were obtained along with eight sequences (~1.3-1.4 kb) equivalent to the begomovirus-associated DNA-satellite components. Four begomovirus sequences obtained from tomato plants were variants of tomato yellow leaf curl virus (TYLCV) with nt sequence identities of 95.3-100%. Additionally, two tomato plants showed a mixed infection of TYLCV and cotton leaf curl Gezira virus (CLCuGeV), okra yellow crinkle Cameroon alphasatellite (OYCrCMA), and okra leaf curl Oman betasatellite (OLCuOMB). Meanwhile, from muskmelon plants, two sequences were closely related (99-99.6%) to the tomato leaf curl Palampur virus (ToLCPalV) DNA-A, whereas two other sequences showed 97.9-100% sequence identities to DNA-B of ToLCPalV, respectively. Complete genome sequences of CLCuGeV and associated DNA-satellites were also obtained from these muskmelon plants. The nt sequence identities of the CLCuGeV, OYCrCMA, and OLCuOMB isolates obtained were 98.3-100%, 99.5-100%, and 95.6-99.7% with their respective available variants. The recombination was only detected in TYLCV and OLCuOMB isolates. To our knowledge, this is the first identification of a mixed infection of bipartite and monopartite begomoviruses associated with DNA-satellites from tomato and muskmelon in Saudi Arabia. The begomovirus variants reported in this study were clustered with Iranian isolates of respective begomovirus components in the phylogenetic dendrogram. Thus, the Iranian agroecological route can be a possible introduction of these begomoviruses and/or their associated DNA-satellites into Saudi Arabia.

Cotton leaf curl Gezira virus

Cotton leaf curl Gezira virus

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.

Association of cotton leaf curl Gezira virus with tomato leaf curl betasatellite infecting Carica papaya in Iran

Papaya leaves exhibiting severe leaf curling and stunting symptoms were tested for the presence of begomovirus infection. The putative full-length cotton leaf curl Gezira virus (CLCuGV) and tomato leaf curl betasatellite (ToLCB) molecules were amplified, sequenced and analyzed. This is the first study providing the identification of CLCuGeV and ToLCB infecting papaya in Iran.

Evidence that leaf curl disease of Malva sylvestris in Iran is associated with cotton leaf curl Gezira virus and associated betasatellite

Evidence that leaf curl disease of Malva sylvestris in Iran is associated with cotton leaf curl Gezira virus and associated betasatellite

New host records for cotton leaf curl Gezira virus: capsicum and melon in Egypt

Cotton leaf curl Gezira virus (CLCuGeV), in association with a DNA-beta satellite, is reported as a causal agent of cotton leaf curl disease. This study reports CLCuGeV detection in field samples of Capsicum annuum and Cucumis melo from Egypt and represents an expansion of the virus host range beyond the Malvaceae family to the two plant families, Solanaceae and Cucurbitaceae.

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.

Diversity, distribution and prevalence of vegetable‐infecting geminiviruses in Burkina Faso

In West and Central Africa, as in many regions of the world, vegetables are severely affected by geminivirus diseases. In Burkina Faso, observation of various virus‐like symptoms, especially on tomato, suggests the involvement of several geminiviruses and underlines the pressing need for additional information on their diversity, distribution, prevalence and host plant reservoirs. Large‐scale surveys conducted in Burkina Faso confirmed the presence of tomato (yellow) leaf curl diseases (ToLCD‐TYLCD) and geminiviruses in all localities with mean prevalences of 25% and 45%, respectively. Five geminiviruses including four begomoviruses (pepper yellow vein Mali virus (PepYVMLV), tomato leaf curl Burkina Faso virus, tomato leaf curl Mali virus and tomato leaf curl Ghana virus) and a dicot‐infecting mastrevirus (chickpea chlorotic dwarf virus) were characterized on tomato. In addition, PepYVMLV and cotton leaf curl Gezira virus (CLCuGeV) were characterized on pepper and okra, respectively, in combination or not with alphasatellites and betasatellites for CLCuGeV. The most severe, prevalent and widely distributed virus on vegetables was PepYVMLV, which was characterized for the first time in combination with a genetically divergent DNA‐B component that may constitute a key factor of PepYVMLV pathogenicity. Of the eight weeds identified as potential reservoir hosts of begomoviruses, four host PepYVMLV. The results confirm the importance of geminivirus diseases on vegetable crops in Burkina Faso and highlight the complex association of geminiviruses and satellites. The detection of begomoviruses in weeds growing close to crops points to the increasing necessity to consider reservoir plants and virus communities in the control of virus diseases.

Identification of a distinct strain of Cotton leaf curl Gezira virus infecting tomato in Oman

AbstractTomato (Solanum lycopersicum) plants exhibiting yellowing, curling and stunting symptoms were identified in fields of the Tawoos Agricultural Systems, in Al‐Batinah in Oman. Cloning and sequencing of restriction endonuclease digested rolling circle amplified viral DNA identified a cotton begomovirus (family Geminiviridae) associated with the symptomatic tomato plants. Detailed analysis of complete sequences showed the virus to be a previously unknown strain of Cotton leaf curl Gezira virus (CLCuGeV) in association with the betasatellite Tomato leaf curl betasatellite (ToLCB). The new CLCuGeV strain, for which the name “Al Batinah” strain is suggested, has the greatest levels of sequence identity (91.9%) to an isolate of CLCuGeV recently reported from the neighbouring United Arab Emirates. Additionally, CLCuGeV‐Al Batinah was shown to have a recombinant origin with sequences donated by an African cassava mosaic virus‐like parent. This is the first identification of this Malvaceae‐adapted begomovirus in tomato. Although ToLCB is common in Oman, being one of only two betasatellites identified there so far, this is the first identification of this betasatellite with CLCuGeV. The significance of these findings is discussed.

Molecular diagnostic development for begomovirus-betasatellite complexes undergoing diversification: A case study

At least five begomoviral species that cause leaf curl disease of cotton have emerged recently in Asia and Africa, reducing fiber quality and yield. The potential for the spread of these viruses to other cotton-vegetable growing regions throughout the world is extensive, owing to routine, global transport of alternative hosts of the leaf curl viruses, especially ornamentals. The research reported here describes the design and validation of polymerase chain reaction (PCR) primers undertaken to facilitate molecular detection of the two most-prevalent leaf curl-associated begomovirus-betasatellite complexes in the Indian Subcontinent and Africa, the Cotton leaf curl Kokhran virus-Burewala strain and Cotton leaf curl Gezira virus, endemic to Asia and Africa, respectively. Ongoing genomic diversification of these begomoviral-satellite complexes was evident based on nucleotide sequence alignments, and analysis of single nucleotide polymorphisms, both factors that created new challenges for primer design. The additional requirement for species and strain-specific, and betasatellite-specific primer design, imposes further constraints on primer design and validation due to the large number of related species and strains extant in ‘core leaf curl virus complex’, now with expanded distribution in south Asia, the Pacific region, and Africa-Arabian Peninsula that have relatively highly conserved coding and non-coding regions, which precludes much of the genome-betasatellite sequence when selecting primer ‘targets’. Here, PCR primers were successfully designed and validated for detection of cloned viral genomes and betasatellites for representative ‘core leaf curl’ strains and species, distant relatives, and total DNA isolated from selected plant species. The application of molecular diagnostics to screen plant imports prior to export or release from ports of entry is expected to greatly reduce the likelihood of exotic leaf curl virus introductions that could dramatically affect the production of cotton as well as vegetable and ornamental crop hosts.

The Prediction of a New CLCuD Epidemic in the Old World.

Cotton leaf curl disease (CLCuD), the most complex disease of cotton, is a major limiting biotic factor to worldwide cotton productivity. Several whitefly-transmitted monopartite begomoviruses causing CLCuD have been characterized and designated as CLCuD-associated begomoviruses. Despite of being reported over 100 years ago in Africa, CLCuD became economically pandemic causing massive losses to cotton production in Pakistan and India during past couple of decades. In Asia, cotton has faced two major epidemics during this period viz. “Multan epidemic” and “Burewala epidemic.” The “Multan epidemic” era was 1988–1999 after which the virus remained calm until 2002 when “Burewala epidemic” broke into the cotton fields in Indo-Pak subcontinent, till 2013–2014. However, both the epidemics were caused by monopartite begomovirus complex. Similarly in Africa, Cotton leaf curl Gezira virus with associated DNA-satellites causes CLCuD. Quite recently, in the Old World (both Asia and Africa), bipartite begomoviruses have started appearing in the areas under cotton cultivation. Under such aggravated circumstances, it seems we are heading toward another epidemic of CLCuD in the Old World. Here we articulate the causes and potential emergence of the third epidemic of CLCuD in Asia. The current situation of CLCuD in Asia and Africa is also discussed.

First reports of Cotton leaf curl Gezira virus and Okra yellow crinkle virus associated with okra leaf curl disease in Côte d'Ivoire

Okra leaf curl disease (OLCD) is commonly observed in okra ( Abelmoschus esculentus ) crops in several African countries (N’Guessan et al ., 1992). Affected plants are severely stunted with apical leaf curl (upward or downward), distortion…

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.

Introduction of Cotton leaf curl Gezira virus into the United Arab Emirates.

Severe leaf curl and small vein thickening symptoms were observed in okra fields in Al-Ain, United Arab Emirates (UAE) during the winter season, 2013. These symptoms were reminiscent of those often associated with begomovirus infection. Based on the symptoms observed in okra plants growing in adjacent fields (20 × 20 m) on two small holding farms, the disease incidence ranged from 90 to 100%. The fields were infested with the whitefly Bemisia tabaci (Genn.), the insect vector of begomoviruses. Total DNA was extracted from four symptomatic okra leaves collected from two plants per field and used for PCR amplification of the core region of the begomovirus coat protein gene using the degenerate primers AVcore 3'-GCCHATRTAYAGRAAGCCMAGRAT-5' and ACcore 3'-GGRTTDGARGCATGHGTACANGCC-5'. Amplicons of the expected size (~579 bp) were cloned and sequenced. BLASTn analysis of the partial coat protein sequences against the NCBI database revealed that the closet match to the four okra isolates was the Cotton leaf curl Gezira virus (CLCuGeV). CLCuGeV is a widespread Old World monopartite begomovirus described from the Nile Basin, sub-Saharan Africa, and southwestern Arabia (1). Recently, this virus has been reported in Jordan (GU945265) and Pakistan (3). To obtain the full-length viral genomic DNA for cloning and sequencing, total DNA extracts were enriched for circular dsDNA by rolling circle amplification (RCA) using Illustra TempliPhi (GE Healthcare, Life Sciences, Piscataway, NJ). The RCA products from each sample were digested with PstI, resulting in ~2.7 and 1.3 kbp molecules, respectively, and cloned into the pGEM plasmid vector (Promega, Madison, WI), linearized with PstI. Two inserts were selected from each cloning event and subjected to DNA sequencing using primer walking. The four resultant sequences of the 2.7 kbp (identified as virus genome) and the 1.3 kbp (betasatellite) inserts shared 99 to 100% nucleotide (nt) identity with each other, respectively. Therefore, only two representative genome and betasatellite sequences of 2,772 bp (KJ939446) and 1,356 bp (KM279620) were deposited in GenBank. Analysis using the Species Demarcation Tool (SDT) (v.1.0) (2) showed that the CLCuGeV UAE isolate sequence shared its highest nt identity (96 to 97%) with isolates from Egypt (AF155064), Pakistan (FR751142), and Jordan (GU945265). In contrast, it was only 93% identical to an isolate of CLCuGeV (HG530540) from the west coast of Saudi Arabia, nonetheless indicating they are all isolates of the same species. Analysis of the CLCuGeV sequence indicated that it was like other monopartite begomoviral genomes, containing a predicted hairpin, REP-binding iterons (GGTACTCA), and a TATA-box in the intergenic region. The genome contained six open reading frames encoding proteins with high homology to other CLCuGeV isolates. The 1,356-bp betasatellite shared its highest nt identity, at 97%, with the Okra leaf curl Oman betasatellite (KF267444) reported from infected okra plants in a neighboring country, Oman. The recent practice of transporting plants between the Gulf countries represents an important means and route for introducing begomoviruses among neighboring countries, compared to the long-distance aerial dispersal of viruliferous whiteflies, which is less likely because the Arabian desert poses a major barrier to long-distance whitefly flights.

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.

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.

Identification of Cotton leaf curl Gezira virus in Papaya in Oman.

Papaya is an important fruit crop in Oman covering some 130 ha with an annual production of 20 tonnes. In 2011, during surveys of farms in the Quriyat region of Oman, papaya plants were found severely affected by leaf curl disease. Leaves with severe curling, vein darkening, and vein thickening were collected for study. Disease incidence ranged from 30 to 50%, particularly in fields with young papaya. A begomovirus (family Geminiviridae) was suspected as the causal agent based on symptoms (1) and the presence of whiteflies in the field. Samples (four to five) were collected from three farms. Total nucleic acids extracted from symptomatic leaves using the CTAB method were used as templates to amplify circular DNAs using Φ29 DNA polymerase and products were digested with restriction enzymes to identify fragments of 2.6 to 2.8 kb typical of geminiviruses. PstI yielded a fragment of ~1.8 kb when the digested product was analyzed by electrophoresis on a 1% agarose gel. The fragment was cloned and sequenced using primer walking strategy in both directions. The sequencing confirmed the exact size (1,764 bp) and the sequence was deposited in GenBank (HE800524). The viral sequence from Oman (isolate Pap-2) showed four open reading frames (ORFs) in the complementary sense (replication associated protein [Rep] gene, the C2 gene, the replication enhancer protein [REn] gene, and the C4 gene) and the virion-sense ORFs (V1 and V2) were missing in the sequence. An initial comparison to NCBI database sequences using BLAST showed the clone from Oman had the highest level of sequence identity to Cotton leaf curl Gezira virus (CLCuGeV) (FJ868828) cloned from okra in Sudan. Subsequent pair wise sequence comparison was done using ClustalV algorithm. Full length sequences of CLCuGeV from database were trimmed according to the size and genomic coordinates of Pap-2 isolate. The Pap-2 isolate sequence was found to have 83.3 to 95.1% sequence identity to CLCuGeV sequences with maximum value to the Sudan isolate. Amino acid sequence comparison showed that the four predicted proteins (Rep, C2, REn, and C4) encoded by the Pap-2 isolate shared 95.3%, 97.8%, 97.7%, and 87.6% sequence identity, respectively, with the homologous proteins of CLCuGeV-SD (FJ868828). The absence of virion-sense protein sequences indicated it to be a subgenomic molecule of CLCuGeV. According to the recommendations of International Committee on Taxonomy of Viruses, these results indicate that the virus identified in association with papaya leaf curl disease in Oman is a variant of CLCuGeV. CLCuGeV is a begomovirus of African origin which is distinct from the begomoviruses of the Middle East and Asia. To our knowledge, this is the first report of CLCuGeV, or any other cotton infecting begomovirus, from papaya in Oman. The presence of a recombinant fragment of CLCuGeV in a Tomato yellow leaf curl virus isolate from Iran (2), and the association of CLCuGeV with cotton in Pakistan (3) and hollyhock in Jordan (GU945265) suggests this virus has moved into the Middle East and Asia from Africa. The identification of CLCuGeV in Oman shows the widespread occurrence of this virus species. This discovery is important since Oman, and other countries in the area, are a hub of international trade and travel, particularly by air and sea, meaning that the virus could spread further.