Molecular characterization of virus isolates from genus Potyvirus infecting Vigna subterranea in Burkina Faso

1 Laboratoire Biosciences, Équipe Génétique et Amélioration des Plantes, Université Ouaga I Pr Joseph KI-Zerbo, 03 BP 7021 Ouagadougou 03, Burkina Faso. 2 Laboratoire de Génétique et de Biotechnologies Végétales, Département de Productions Végétales, Institut de l’Environnement et de Recherches Agricoles (INERA), 04 BP 8645 Ouagadougou, Burkina Faso. 3 Laboratoire de Virologie et de Biotechnologies Végétales, Département de Productions Végétales, Institut de l’Environnement et de Recherches Agricoles (INERA), 01 BP 476, Ouagadougou, Burkina Faso. 4 Institut de Recherche pour le Développement (IRD), 911 Avenue Agropolis, 34394 Montpellier, France. 5 Laboratoire Mixte International Patho-Bios, 01 BP 476, Ouagadougou, Burkina Faso.


INTRODUCTION
Bambara groundnut [Vigna subterranea (L.) Verdc.] is an indigenous legume that originated from Africa precisely in the northern part of Nigeria and Cameroun (Goli et al., 1997).It is mainly grown for human consumption and plays an important socio-economic role in tropical Africa (Nadembega, 2016).Its seeds contain an average of 63% carbohydrate, 19% protein and 6.5% oil (Mkandawire, 2007).This well balanced composition makes it a complete food, thus Bambara groundnut could be used to alleviate nutritional problems especially for the rural population (Bamshaiye et al., 2011).In Burkina Faso, it is ranked second seed legume after cowpea [Vigna unguiculata (L.) Walp] in terms of production and consumption (Ouédraogo et al., 2008).
Among these viruses found to occur on bambara groundnut, only four have been reported from Burkina Faso using serological and biological tests.They include CABMV, BCMV-BlCM, PeMoV and CPMoV (Sérémé, 1989;Drabo et al., 1997;Néya, 2011).However, none of the studies have molecular characterized bambara groundnut viruses from this country.Whereas, improving our knowledge in the virus characterization would help to establish some effective control strategies against bambara groundnut viral diseases (Frenkel et al., 1992).
In this study, serological and molecular detection tests were used to screen for the presence of viruses within bambara groundnut leaves samples collected in the Sudan (humid), Sudan-Sahel (sub-humid), and Sahel (dry) agro-climatic zones of Burkina Faso.Virus identified was further characterized for their phylogenetic relationship with GenBank viruses.

Plant sampling
During the months of September to October of the year 2016, a sampling was carried out on bambara groundnut fields.Symptomatic leaves showing leaf curling, stunting and mosaic diseases were sampled in the three agro-climatic zones of Burkina Faso.Sampling concerned farmers' fields and experimental plots.A total of 140 samples were collected with 5 in the sahel zone, 125 in the sudan-sahel zone and 10 in the sudan zone.Leaves collected were placed on melting ice before storage at -80°C.Each collected sample was divided into three parts.The first was used for double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) serological tests, the second for reverse transcriptionpolymerase chain reaction (RT-PCR) molecular tests and finally the third part was retained as eventual inoculum.

Serological detection test
The presence of viruses CABMV, BCMV-BlCM, CPMoV, CPMV and PeMoV in the samples collected were detected by using their corresponding polyclonal antibodies from ELISA detection kits.ELISA detection kits were purchased from AC Diagnostics, Inc. (USA) and a DAS-ELISA (Clark and Adams, 1977) was performed following the manufacturer's protocol.

RNA extraction, RT-PCR and sequencing
For molecular characterization, RNA was extracted from collected samples leaves using Trizol reagent (Invitrogen, USA) extraction protocol as described in Longué et al. (2017).
The cDNAs were constructed in two steps.Firstly, 5 µl mixture of total RNA and oligo dT (10 µM) were incubated at 70°C for 5 min and immediately placed on ice for 5 min.Secondly, a mixture constituted of 1.25 µl of dNTPs (10 mM), 5 µl of M-MLV RT Buffer (5x) (Promega, Corp. USA), 0.5 µl of enzyme M-MLV RT RNase (200 U) (Promega, Corp. USA) and H2O qsp was added to the previous 5 µl mixture.Then, the total volume of 25 µl was incubated at 40°C for 1 h followed by 70°C for 15 min to generate cDNA.
The degenerated primers pair P077/P078 (Table 1) (Marie-Jeanne et al., 2000) was used for further identification of Potyvirus in the samples.To sequence the Potyvirus (CABMV and BCMV-BlCM), the whole coat protein gene, primers P105 and P106 were designed based on GenBank available sequences.Then, primers combination P105/P078 and P077/P106 (Table 1) were used in independent PCR.Amplification products were separated by electrophoresis on a 1% agarose gel containing ethidium bromide and then visualized under UV light.Expected amplicons for 12 samples were direct sequenced with forward and reverse primers using the Sanger method (GENEWIZ, UK).
Phylogenetic tree was constructed based on 476 nt obtained from 12 samples.Sequences were aligned using MUSCLE (Edgar, 2004) with default setting.Maximum likelihood phylogenetic tree was performed in MEGA 6.0 (Tamura et al., 2013) using the Tamura Nei parameter (TN93+G+I) nucleotidic substitution model.Bootstrap method at 1000 replicates was adopted to support the branches.
The pairwise nucleotide identities were performed using SDT software version 1.2 (Muhire et al., 2014).Sequences obtained in this study have been submitted to Genbank (Table 3).

Virus detection in DAS-ELISA and RT-PCR tests
DAS-ELISA was efficient to identify only CABMV and BCMV-BlCM from the genus Potyvirus, respectively in 7.14 and 1.43% samples to make a total of 8.57% (12/140) positive samples (Table 2).None samples was detected positive to CPMoV, CPMV and PeMoV antibodies.However, RT-PCR tests were more efficient in Potyvirus detection (14.29%, 20/140) using primers pair P077/P078 (Marie-Jeanne et al., 2000).Beside positive samples in DAS-ELISA, eight new samples become positives (Table 2).Figure 1 shows the detection  (476-950 nt) revealed that nine samples were identified to CABMV Genbank accession number KT726938 at 88 to 98% identity (Table 3) whereas, three samples were identified at 97 to 98% identity to BCMV Genbank accession number AJ312438 (Table 3).These confirmed the identification of CABMV and BCMV-BlCM in DAS-ELISA test.

Phylogenetic and nucleotide identity analyses
The maximum likelihood phylogenetic tree (Figure 3) showed that the 5' partial coat protein sequence (476 nt) was sufficient to separate the two Potyvirus CABMV and BCMV-BlCM.
All Burkina Faso CABMV isolates clustered together with Uganda isolate (KT726938).However, they might form two distinct groups.The first one is most closed to Uganda isolate (KT726938) at bootstrap 86%.Members of this group shared together 99.6 to 100% identity and 97.5 to 97.7% identity to Uganda strain (Table 4).
Members of the second group showed in an isolate group and shared together 83.4 to 99.4% identity.However, pairwise identities between Uganda isolate (KT726938) and members of the second group (85.1 to 93.3%) (Table 4) were low.Furthermore, identities the two groups of CABMV ranged from 84.9 to 92.4%.
In Burkina Faso, all CABMV isolates and Uganda isolate nested to the Nigeria isolate (Y17822) at bootstrap 97 (Figure 3).

DISCUSSION
Several viruses have been reported to infect bambara groundnut (Thottappilly and Rossel, 1997).This study identified only Potyvirus CABMV and BCMV-BlCM infecting bambara groundnut in Burkina Faso both in DAS-ELISA and RT-PCR tests.This may be associated to the high prevalence of these viruses (over 65%)    3).Bootstrap method was adopted at 1000 replicates.
reported in the country on cowpea crop (Néya, 2011;Palanga et al., 2016).Bambara groundnut and cowpea are two legume crops cultivated under the same climatic conditions and sometime in association.However, their prevalence in DAS-ELISA (8.57%) was less than in RT-PCR (14.29%).Indeed, some specificity in the coat protein structure of some virus strain may result in their false detection in serological test, whereas primers used in RT-PCR were specific and degenerated (Table 1) to amplified maximum of CABMV and BCMV-BlCM strains.
On the other hand, some studies reported the efficiency of ELISA tests in plant virus detection (Konaté and Néya, 1996;Akinjogunla et al., 2008;Lima et al., 2012), but Gillaspie et al. (1999), Sipahioğlu (2005) and Liebenberg et al. (2009) showed that PCR and RT-PCR molecular tests were more sensitive.Sudan zone (humid) was observed to be the most infected (40%) than sudan sahel (sub humid) (12.8%) and sahel (dry) (0%).Thus, climate might have an influence on Potyvirus infection and distribution in bambara groundnut crop.Indeed, Dabiré (1992) and Néya et al. (2008) also showed that the propagation of CABMV epidemic in cowpea crop and aphid population (vectors of these Potyvirus) decreased from the sudan zone to the sahel.Elsewhere, Estay et al. (2009) reported that aphids' population increase is a key factor to the spread of viruses and it depend on climate.In other parts, the prevalence of CABMV in cowpea was also found higher in the sudan than the sudan-sahel and the sahel (Néya, 2011;Palanga et al., 2016).
However, this study reports that CABMV was most prevalent than BCMV-BlCMV even in DAS-ELISA (7.14 against 1.43%, respectively) and RT-PCR (10.71 against 2.86%, respectively).This is in agreement with the study of Néya (2011) and Palanga et al. (2016) work on cowpea.
The none identification of CPMV, CPMoV and PeMoV might explain their absence in the 140 bambara groundnut samples tested.However, Sérémé (1989) had already reported CPMoV and PeMoV on bambara groundnut in the country.
The phylogenetic tree supports the occurrence of the two Potyvirus species CABMV and BCMV-BlCM on bambara groundnut in Burkina Faso.The specificity of nucleotide sequence to distinguish these two viruses was reported by Grisoni et al. (2006) and Palanga et al. (2016).Two groups within Burkina Faso CABMV isolates were observed on the phylogenetic tree (Figure 3).When the first group (MF277036, MF277033, MF277035, MF277037, MF277039) shared high nucleotide identity (97.5 to 97.7%) with Ugandan isolate (KT726938), the second group (MF277031, MF277034, MF277038, MF277041) was at 85.1 to 93.3% identity to the same Ugandan isolate.According to the Potyvirus strain criteria demarcation proposed by Shukla and Ward (1988) (with nucleotides sequence identity >95% in the coat protein), members of the first group and the Ugandan isolate (KT726938) might be the same strain.However, member of the second group might still be an isolate strain.These results might support the presence of two strains of CABMV infecting bambara groundnut in Burkina Faso.Indeed, previous study based on serological test revealed the presence of four serotypes I, II, III and IV within CABMV species infecting legume crops in Burkina Faso (Néya, 2011).Beside the Ugandan isolate (KT726938), all Burkina Faso CABMV isolates were closed to Nigerian isolate (Y17822) (78.4 to 84.5% identity).
The high seed-transmission of CABMV on cowpea (3 to 100%) was shown by Konaté and Néya (1996), Néya (2002) and Barro et al. (2016).It was considered to be the first source of infection to initiate CABMV epidemic (Néya et al., 2007).Bambara groundnut or other legume crops seeds trade between countries or seeds exchange between research centers could explain the strong relationships between Uganda and Burkina Faso isolates.
Altogether, our study reports the first molecular characterization of virus from genus Potyvirus in bambara groundnut from Burkina Faso.

Conclusion
This study reveals the occurrence of CABMV and BCMV-BlCMV infecting bambara groundnut in Burkina Faso.However, CABMV was the most prevalent.Beside, RT-PCR was the most accurate tool for these virus detection in bambara groundnut; the phylogenetic analyses permit the understanding of evolutionary relationships between Burkina Faso isolates and others Genbank available species.However, further analysis based on the whole coat protein gene in amino acid or nucleotide and increase of samples number is required for better characterization of virus from genus Potyvirus occurring in bambara groundnut fields in Burkina Faso.

CONFLICT OF INTERESTS
The authors have not declared any conflict of interest.
)76003305.Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License

Figure 3 .
Figure 3. Maximum likelihood phylogenetic tree based on 476 nt in coat protein gene showing relationship between Burkina Faso isolates of CABMV and BCMV-BlCM (MF277031 to MF277042) and Genbank related species (Table3).Bootstrap method was adopted at 1000 replicates.

Table 1 .
List of primers used in this study.
Single letter code: H = A/C/T; Y = C/T; R = A/G; K = G/T; Ta, annealing temperature; Ext, extension time.

Table 2 .
Potyvirus detection in DAS-ELISA and RT-PCR according to sampling area.

Table 3 .
List of virus isolates from the genus Potyvirus from Burkina Faso and some GenBank accessions analyzed in this study.

Table 4 .
Percentage of nucleotide identity between Burkina Faso isolates of cowpea aphid-borne mosaic virus (CABMV) and blackeye cowpea mosaic virus strain of bean common mosaic virus (BCMV-BlCM) and Genbank related sequences based on 476 nucleotides in the coat protein.