The most damaging and economically important diseases of crops, especially in tropical and subtropical regions are caused by the whitefly-transmitted begomoviruses. These viruses are included in the genus Begomovirus of the family Geminiviridae and are responsible for causing crop losses ranging from 30% to 100%  . Begomoviruses are single-stranded circular DNA viruses, which replicate in the nucleus of their host cells and are classified in monopartite or bipartite viruses depending on their genome organization  . They have emerged everywhere in the world where environmental conditions support large whitefly (Bemisia tabaci Genn.) populations and in Western Africa, emergence of begomoviruses is caused by genetically distinct species that have evolved locally   .
Cassava mosaic disease (CMD) caused by whitefly-transmitted begomoviruses is the main factor of cassava (Manihot esculenta Crantz) decrease yields  . Eight cassava mosaic begomovirus species have been reported in Africa    of which three species affect cassava in Togo: ACMV (African cassava mosaic virus), EACMV (East African cassava mosaic virus) and ICMV (Indian cassava mosaic virus)  . Begomoviruses are also reported in pepper (Capsicum sp.)    which can be intercropped with cassava. Pepper is an important cash crop for smallholder farmers in developing countries. Among the five cultivated species of the genus Capsicum, Capsicum annuum (both hot and sweet pepper) is the most widely cultivated. In cassava production, pepper is often intercropped with it in Togo.
This study was initiated to identify cassava begomoviruses on other crops in cassava intercropping systems. Results of this study will help to better understand the disease epidemics, related to whitefly-transmitted begomoviruses as well as Bemisia tabaci biotypes behavior within these systems.
2. Material and Methods
2.1. Foliar Sampling and DNA Extraction
Foliar samples from pepper plants intercropped with cassava showing typical symptoms of viral diseases (mosaic, distortion, leaf curling, yellowing and deformation) were collected through the five economic regions of Togo.
Total DNA was extracted from collected leaves using the DNA minipreparation method  .
2.2. PCR Diagnosis
Detection of cassava mosaic begomoviruses (CMBs) in samples was performed by PCR amplification using three sets of specific primers targeting the coat protein (Table 1): JSP001/JSP002, JSP001/JSP003 and JSP012/JSP013 allow to identify respectively African cassava mosaic virus (ACMV), East African cassava mosaic virus (EACMV) and Indian cassava mosaic virus (ICMV)  . Another PCR was performed using begomoviruses coat protein specific primers AC1048/AV494  to identify other begomoviruses apart from CMBs. PCR tests were done in the thermocycle “Mastercycle gradients Eppendorf (Hamburg, Germany). All PCR reactions were prepared in a 25 µl reaction mixture containing: template DNA 2 µl, dNTPs 0.5 µl (200 µM), primers 1.25 µl (each), Taq polymerase 0.16 µl (0.8 U), reaction buffer 2.5 µl (1X), MgCl2 2.5 µl (2.5 mM), BSA 0.5 µL (0.4 µg/µL), and Sterile water 14.34 µl.
Table 1. Primers used in this study.
D = A, G, T; H = A, C, T; K = G, T; M = A, C; N = A, C, G, T; R = A, G; W = A, T; Y = C, T.
The PCR thermal profile for CMBs was as followed: a 94˚C denaturation step of 2 min followed by 30 cycles of denaturing at 94˚C for 30 secs, annealing at (50˚C for ACMV and EACMV and 52˚C for ICMV) for 30 secs and extension at 72˚C for 1 min and then a final extension step at 72˚C for 10 min  . The PCR thermal profile with primers AC1048/AV494 was: a 94˚C denaturation step of 2 min followed by 35 cycles of denaturing at 94˚C for 1 min, annealing at 55˚C for 2 min and extension at 72˚C for 1 min and then a final extension step at 72˚C for 10 min  .
PCR amplified products were resolved by agarose gel electrophoresis and visualized under ultraviolet (UV) light using Gel Doc 2000 (Bio-Rad, Hercules, CA, USA). A 100 bp DNA molecular weight marker (Smart ladder small fragments Eurogentec) was run in each gel as a reference to estimate the size of the virus-specific DNA band in the PCR amplified products.
2.3. DNA Sequencing and Phylogenetic Analysis
Illumina Nextera XT Index kit (Illumina Inc., San Diego, CA, USA) was used according to the manufacturer’s instructions to assign a code to each sample before the sequencing. Purity of the PCR products was verified with the Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA, USA) and the sequencing was performed at the UPJV Molecular Biology platform based on “Sequencing by Synthesis” technology. Illumina Miseq Reagent Kit V2 (Illumina Inc., San Diego, CA, USA) was used to perform this sequencing.
BLASTn program (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used for preliminary analysis  . Multiple sequence alignments were performed with ClustalX 2.1  and pairwise nucleotide identities were calculated with SDT 1.2 software  .
Phylogenetic analysis was performed by Neighbor-Joining method using Darwin6. A thousand bootstrap replications, was used to evaluate the robustness of each individual branch. Tree was visualized and edited using Darwin6.
The nucleotide sequences of the following begomovirus (GenBank accession numbers are given) were included in the analysis: African cassava mosaic virus (AF259894.1, AY562423.1, EU685318.1, FM877473.1, HE979761.1, HG530111.1, KJ887779.1, KR476371.1, KR476372.1); East African cassava mosaic virus (AJ717553.1, HG530116.1); East African cassava mosaic Cameroon virus (AF112354.1, KJ887944.1); East African cassava mosaic Kenya virus (JF909078.1, KJ888087.1); East African cassava mosaic virus-Tanzania (Z82356.1); East African cassava mosaic virus-Uganda (KT780440.1); Ageratum leaf curl Cameroon virus (FR717144.1, FR873228.1, FR873230.1); Pepper yellow vein Mali virus (KY271076.1, FM876851.1, AM691555.1, AY502935.1) and Pepper golden mosaic virus (EF027236.1).
3.1. Incidence Rate of Begomoviruses
A total of nineteen samples were collected (Table 2). Samples number was varied from one region to another, most of them were collected in Maritime and Plateaus regions, which are the main cassava production areas in Togo. In Kara region, during the collection, pepper was not found in association with cassava (Figure 1).
Figure 1. Geographical location of the sample sites.
PCR products with the expected size were amplified from collected samples with primers JSP001/JSP003 and AC1048/AV494 confirming the association of begomovirus with symptoms. This indicates in addition, that pepper is natural infected by East African cassava mosaic virus (EACMV). Based on PCR results with primers JSP001/JSP003 and AC1048/AV494, 10.53% and 26.31% of samples were respectively infected (Table 2). African cassava mosaic virus and Indian cassava mosaic virus were not identified in none sample respectively with primers sets JSP001/JSP002 and JSP012/JSP013.
3.2. Sequence Analysis and Comparison
PCR products were submitted to direct sequencing. Three sequences obtained from these products were selected for phylogenetic and pairwise nucleotide analysis
Table 2. Incidence rate of begomoviruses.
- = Sample reacted negatively to PCR; + = Samples reacted positively to PCR.
(Table 2). The criteria used to distinguish between different viruses are: 90% - 100% for isolates, 80% - 90% for strains and less than 80% for species demarcation  .
Sequence comparisons revealed that isolate B51 from primers JSP001/JSP003 specifics to East African cassava mosaic virus (EACMV), shared the highest sequence identity (74%) with EACMV AJ717553.1; but also, with African cassava mosaic virus (ACMV) KJ887779.1 and Pepper yellow vein Mali virus (PepYVMV) isolates AM691555.1, AY502935.1 and KY271076.1 (Figure 2). Thus, the new strain of EACMV found in pepper seemed to be related to ACMV and PepYVMV species. This finding is supported by the phylogenetic analysis which showed isolate B51 forming a distinguish group with isolate G44a. Both isolates were closely related to group of Ageratum leaf curl Cameroon virus (ALCCMV) (Figure 3).
Isolates G44a and G44b from primers AC1048/AV494 shared high level of nucleotides identity respectively with Ageratum leaf curl Cameroon virus (ALCCMV) isolate FR873230.1 (80%) and ACMV AF259894.1 (93%). Isolate
Figure 2. Pairwise nucleotide identities scores between sequences of isolates B51, G44a, G44b and selected begomovirus from GenBank.
Figure 3. Phylogenetic tree based upon an alignment of isolates B51, G44a, G44b and selected begomovirus sequences from GenBank.
G44b also shared 90% identity with EAMCV references (HG530116.1 and KT780440.1) (Figure 2). These results show that isolate G44a is an Ageratum leaf curl Cameroon virus and isolate G44b would be a recombinant of African cassava mosaic virus and East African cassava mosaic virus. In the phylogenetic tree, isolate G44b formed a distinguish group (Figure 3).
This study has demonstrated three unknown infections of begomovirus in pepper foliar samples including Ageratum leaf curl Cameroon virus (ALCCMV), a new strain of East African cassava mosaic virus (EACMV) and a recombinant of African cassava mosaic virus (ACMV) and EACMV.
Diagnosis of cassava mosaic begomoviruses (ACMV and EACMV) in pepper showed that these begomoviruses could infect other crop species than cassava. This is the first identification and characterization of begomoviruses responsible of cassava mosaic disease in another cultivated plant in Togo. The ability of begomoviruses to infect plants belonging to different taxonomic families has been studied. Indeed, Tomato yellow spot virus (ToYSV) can infect tomato (Solanaceae) and soybean (Fabaceae); likewise, Tomato leaf curl New Delhi virus (ToLCNDV) infects Solanaceae and Cucurbitaceae  .
It is known that genomic plasticity of begomoviruses allows them to adapt to new environments and hosts  and results found here suggest the presence of polyphagous biotypes in populations of Bemisia tabaci vector of these viruses which can colonize with efficiency cassava and pepper plants. In addition, climate changes directly affect insect behavior, fertility, development and survival or indirectly affect insect by changes in trophic relationships   . Pepper samples were all collected in cassava intercropping systems, which means that in fields where cassava and pepper plants were both present. Hence, the polyphagous biotype could easily visit infected cassava plants and transmit cassava virus to pepper plants by feeding on them.  found that the emergence of new begomoviruses might be occurring in relationship with some factors like evolution of new virus variants, the appearance of efficient vectors, changes in intercropping systems, introduction of susceptible plant varieties, and changes in climatic conditions. Isolates G44a and G44b found here were both recombinants.
A general consensus exists that Bemisia tabaci is a complex of morphologically indistinguishable populations with different biological biotypes. The cassava biotype of Bemisia tabaci is known to colonize only cassava and wild eggplant whereas the sweet potato biotype colonizes various plant and weed species but not cassava  . But recent work showed that, the sweet potato biotype of Bemisia tabaci can colonize cassava. In fact, different species of Bemisia tabaci were reported to have cassava and other crops together as host plants  . In particular, SSA3 species was found on cassava, cotton, eggplant, groundnut and tomato  . In the same way, MED-Q1 adults, larvae, and nymphs were collected on cassava  . The capacity of biotype B to adapt gradually to cassava was studied by  . These authors found that the biotype B of the insect adaptation started on Phaseolus vulgaris, followed on Euphorbiaceae plants until finally reaching a commercial cassava variety.
In cassava intercropping systems, these intermediate hosts or plants belonging to the same family are used as secondary crops associated with cassava and weeds can belong to Fabaceae and Euphorbiaceae families.  showed that ACMV and EACMV infect naturally other plant species than cassava namely Senna occidentalis (L.), Leucaena leucocephala (Lam.), Combretum confertum (Benth.) and Manihot glaziovii (Müll.Arg). Apart from wild cassava (M. glaziovii),  also identified EACMV on Albizia zygia (DC.), Senna obtusifolia (L.), Pupalia lappacea (L) and Strophanthus hispidus (DC.). Furthermore, Uganda variant EACMV-UG has been detected in a Jatropha curcas (L.)  . However, it is important to note that none of these authors had identified EACMV (East African cassava mosaic virus), in another crop outside cassava.
Ageratum leaf curl Cameroon virus was first identified on Ageratum conyzoïdes (L.) in Cameroon  and reported in Togo on tomato  . The identification of this begomovirus on pepper now approved that this begomovirus can infect more than one host including weeds as well as cultivated plants.
Occurrence of new begomovirus species on pepper could lead in case of mixed infections with already known begomoviruses infecting this crop to recombinant actions    . This situation might result in increasing begomovirus emergence in cassava intercropping systems.
This study showed that cassava mosaic begomoviruses could infect other cultivated plants than cassava and suggested a change in Bemisia tabaci population or in its feed habit. Further investigations will bring more information about cassava mosaic begomoviruses and pepper relationships.
This work was supported by International Foundation of Science (IFS) research grant C/5596-1 to K.A. Dansou-Kodjo; and by West African Virus Epidemiology for Togo (WAVE-UL) project.