Occurrence of Cassava Mosaic Begomovirus New Species and Ageratum Leaf Curl Cameroon Virus on Pepper (Capsicum annuum L.) in Togo

Cassava mosaic disease caused by the whitefly-transmitted begomoviruses (family Geminiviridae) is a major threat to cassava (Manihot esculenta Crantz) production, which can be intercropped with other plants such as pepper (Capsicum annuum L.). The aim of this study is to identify cassava begomoviruses on other crops in cassava intercropping systems. Thus, foliar samples showing typical symptoms of virus diseases in cassava intercropping systems were collected from pepper and submitted to PCR analysis and direct sequencing. Three begomovirus species ACMV, EACMV and ALCCMV were identified and characterized in samples. Isolates of these species shared respectively 90% 93%, 74% and 80% nucleotide identities with begomoviruses. These findings show that cassava begomoviruses can infect other crops and will help in understanding the epidemiology related to whitefly-transmitted begomoviruses in cassava intercropping systems.


Introduction
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 cate in the nucleus of their host cells and are classified in monopartite or bipartite viruses depending on their genome organization [2]. 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 [3] [4].
Eight cassava mosaic begomovirus species have been reported in Africa [6] [7] [8] 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) [8]. Begomoviruses are also reported in pepper (Capsicum sp.) [9] [4] [10] 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.

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 [11].

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) [12]. Another PCR was performed using begomoviruses coat protein specific primers AC1048/AV494 [13] 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.   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 [12]. 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 [13].
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.

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).

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     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).
It is known that genomic plasticity of begomoviruses allows them to adapt to new environments and hosts [19] [23]. 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 [24]. In particular, SSA3 species was found on cassava, cotton, eggplant, groundnut and tomato [25]. In the same way, MED-Q1 adults, larvae, and nymphs were collected on cassava [26]. The capacity of biotype B to adapt gradually to cassava was studied by [27].  [29] 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.) [30]. 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 [31] and reported in Togo on tomato [32]. The identi-

Conclusion
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.