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

Fidle Tiendrbogo 0 1 Pierre Lefeuvre 0 Murielle Hoareau 0 Julie Villemot 0 Gnissa Konat Alfred S Traor 1 Nicolas Barro 1 Valentin S Traor Bernard Reynaud 0 Oumar Traor Jean-Michel Lett 0 0 CIRAD, UMR 53 PVBMT CIRAD-Universite de la Reunion, Pole de Protection des Plantes , 7 Chemin de l'IRAT, 97410 Saint Pierre, La Reunion , France 1 Laboratoire de Biochimie & Biologie Moleculaire, CRSBAN/UFR/SVT , Universite de Ouagadougou 03 BP 7021 Ouagadougou 03 , 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. - Findings Okra leaf curl disease (OLCD) is commonly observed among okra (Abelmoschus esculentus) crops in Burkina Faso (BF) and several African countries [1-5]. Affected plants are severely stunted with apical leaf curl (upward or downward), distortion and thickening of the veins. In BF, okra is widely grown in both rainy and dry seasons. It is a major source of income particularly for smallscale farming. Viral diseases are important constraints in the production of this crop [6]. Recently, it was shown that OLCD in Africa is associated with a complex of begomoviruses: Cotton leaf curl Gezira virus (CLCuGV; [7,4,5]), Okra yellow crinkle virus (OYCrV; [8]) and Hollyhock leaf crumple virus (HoLCrV;[9,10]). Viruses of the genus Begomovirus belong to the family Geminiviridae and are transmitted by the whitefly vector Bemisia tabaci to dicotyledonous plants [11]. They have emerged as a major constraint for many vegetable and fibre crops throughout the world [12]. Begomoviruses are either bipartite with two genomic components, designated as DNA-A and DNA-B or monopartite with only DNA-A like components [13]. Some of the monopartite begomoviruses are also associated with additional circular ssDNA molecules, such as betasatellite or alphasatellite (previously known as DNA-1) that are nearly half the size of DNA-A. Betasatellites have been involved in pathogenicity but alphasatellites have no known function and are certainly not involved in symptom induction [14-16]. Alphasatellites have only been shown to be present in plants infected with monopartite begomoviruses in association with betasatellites [17]. The aim of our study was to characterize at the molecular level the complex of viruses involved in OLCD in BF and their relationship with other begomoviruses. In association with a single Old World begomovirus, we describe their associated satellite DNAs. During May 2008 to April 2009, 74 leaf samples exhibiting typical OLCD symptoms were collected from okra fields in the major growing regions of BF around Tibl, Kampala, P, Kamboins, Bazga and Bama (Kou valley) localities. Total DNA was extracted using DNeasy Plant Minikit (Qiagen) before detection of begomoviruses using polymerase chain reaction (PCR) with specific primers of either the DNA-A [18] or betasatellite and alphasatellite [19,20]. Full-length viral genomes were amplified from the PCR-positive samples by rolling-circle amplification (RCA) [21]. The amplified DNAs were digested with endonucleases BamHI or PstI, and the DNA fragments of the expected size (~2.8 kb for DNA-A and ~1.4 kb for satellites) were cloned into pGEM-3Zf (+) vector (Promega Biotech). Cloned genome components were sequenced by Macrogen Inc. (South Korea). Contigs were assembled with the DNAMAN software (Lynnon, Quebec, Canada) and subsequently aligned using the ClustalW tool [22] implemented in MEGA 4 [23]. Sequence comparisons were performed in MEGA 4 with pairwise deletion of gaps. The optimal model of sequence evolution, defined with ModelTest [24], was used for maximum likelihood (ML) phylogenetic reconstruction using PHYML_v2.4.4 [25]. The degree of support for individual branches within the resulting phylogenetic trees was assessed with 1000 full ML bootstrap iterations. The trees were visualized using FigTree v1.1.1 software. Recombination was analyzed using our sequences and a set of sequences representing the whole African begomovirus diversity (representing an alignment of 121 sequences). Detection of potential recombinant sequences, identification of likely parental sequences, and localization of possible recombination breakpoints was carried out using RDP [26], GENECONV [27], BOOTSCAN [28], MAXIMUM CHI SQUARE [29], CHIMAERA [28], SISTER SCAN [30] and 3Seq [31] recombination detection methods as implemented in RDP3 [32]. The analysis was performed with default settings for the different detection methods and a Bonferroni corrected P-value cut-off of 0.05. Only events detected with 3 methods or more were accepted. Despite a very poor preservation of samples (high necrosis), 48 samples of the 74 were detected as being infected with begomovirus using PCR amplifications with the universal primer pair VD360-CD1266 recovering the conserved CP ORF [18]. From the positive samples, 23 begomovirus genome sequences with length between 2761 to 2773 nucleotides (nt) were successfully obtained using RCA. Pairwise sequence comparison demonstrated that the 23 new genome sequences of monopartite begomoviruses from BF are genetically related to the same strain (94.7 to 100% identity amongst themselves). A BLAST search identify the most similar virus sequences as being Cotton leaf curl Gezira virus (CLCuGV) isolates, a monopartite Begomovirus first identified in Sudan [33]. Further pairwise sequence analyses showed that the 23 sequences shared between 94.8 to 98.8% nt identity with CLCuGV isolates from Niger (FJ469626, EU432373, EU432374), 93.7 to 96.2% with CLCuGV from Sudan (AY036007, AY036008), 92.4 to 96.1% with CLCuGV from Egypt (AY036006, AY036010) and 89.3 (...truncated)