The first report of bean common mosaic virus (BCMV) infection of African yam bean (Sphenostylis stenocarpa) in Nigeria

African yam bean (Sphenostylis stenocarpa) is an underutilized crop that has the potential to contribute to sustainable food security. In October 2021, more than 90% African Yam Bean (AYB) plants showed typical virus symptoms of mosaic and necrosis in the grain legumes field of the Institute of Agricultural Research and Training (IAR&T), Nigeria. Subsequently, leaf samples were collected and tested by ELISA and PCR to identify the virus species. Anti-BCMV and anti-potyvirus antibodies both gave positive results when symptomatic leaves were tested, and PCR using primers designed to the coat protein gene of BCMV amplified a band of the expected size (469 bp). The sequence of the PCR product was deposited in GenBank with the accession No. OL763314. The nucleotide sequence of the coat protein gene had 99% identity with BCMV isolate TN2 (KY044818). The identities of the nucleotide and amino acid sequence of the partial CP gene of the isolated virus relative to those of other potyviruses were 82.96–99.12% and 87.33–100%,, respectively. Phylogenetic analyses of the partial CP-nucleotide sequences grouped the isolate from this study (BCMV-IART-AYB) and BCMV-TN2 in the same cluster with other BCMV strains of the peanut stripe (PSt) and the blackeye cowpea (BlC) strains. In this study, we identified Bean commom mosaic virus (BCMV) infecting AYB for the first time in Nigeria and show that it has high nucleotide and amino acid identity with an Isolate of cowpea-infecting BCMV in India and China respectively than isolate in Nigeria.


Introduction
African Yam Bean (Sphenostylis stenocarpa) is an underutilized crop that is gaining attention as a crop that could contribute to sustainable food security due to its benefits of producing both nutritious leguminous grains and tubers in single plants [1,2]. African Yam Bean (AYB) has been reported to produce grains and tubers which have protein more than 2 times the amount in potato and higher than in yam and cassava and amino acid value higher than the value in pigeon pea, cowpea, and bambara groundnut [3].
African Yam Bean (AYB) is mostly grown in the Central and West African regions and most extensively in Nigeria [4]. Being a previously underutilized crop, research has focused on exploring the genetic enhancement in breeding programs focused mainly on yield and nutrient content. Another important consideration is the association with pests and disease that may play a significant role in in reducing productivity. In Nigeria, several viral and fungal diseases associated with AYB have been documented [5][6][7]. Bean common mosaic virus in the genus Potyvirus is known to infect many leguminous species including cowpea and soybean [8] and transmission is predominantly through seeds. However, the aphid species Aphis fabae and Myzus persicae can also transmit the virus in a nonpersistent manner [9,10]. Previously, Ogunsanya et al. [7] in their study to screen 20 accessions of AYB for resistance to viral diseases reported that AYB was susceptible to two viruses, namely, cowpea mild mottle Virus (CPMMV) and blackeye cowpea mosaic virus (BlCMV) both of which have been detected using ELISA methods in Nigeria. However, there has not previously been reports of BCMV infecting yam bean in Nigeria.

Materials and methods
In October 2021, more than 90% African Yam Bean (AYB) plants showed typical virus symptoms of mosaic and necrosis in the grain legumes field of the Institute of Agricultural Research and Training (IAR&T), Nigeria (Fig. 1). Leaf samples were collected from symptomatic and asymptomatic plants (N = 25). The samples were tested using ELISA following the manufacturers protocols. To further confirm virus identity, selected samples from the plants that gave positive results following ELISA were subjected to PCR using primers designed to the coat protein as follows BCMV F1: 5′-ATG TGG TAC AAT GCT GTG AAG-3′/BCMV R1: 5′-TTT CAG TAT TCT CGC TGG T-3′. Total RNA was extracted from AYB leaf samples using modified CTAB method [11]. The extracted RNA was used as template with in reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify 469 bp of the coat protein (CP) gene of BCMV. The RT-PCR consisted of a RT-Phase of 44 °C for 30 min then each cycle in PCR consisted of initial denaturation at 94 °C for 1 min,35 cycles of denaturation at 94 °C for 60 s, annealing at 54 °C for 60 s, extension at 72 °C for 60 s, and a final extension at 72 °C for 10 min. Products were visualized on a 1.2% (w/v) tris-acetate agarose gel stained with ethidium bromide. Two independent successfully amplified RT-PCR products were sent for Sanger sequencing in both direction (Inqaba biotec, Pretoria, South Africa) using the amplificiation primers. The nulceotide sequence of one of the isolates was submitted to NCBI GenBank. A phylogenetic tree was constructed by first aligniing sequences using Muscle (MEGA 7.0, [12]) with the neighbor-Joining method and bootstrap support was estimated by resampling the data 1,000 times.

Results and discussion
Based on the high disease incidence and the symptom severity (Fig. 1), it is probable that BCMV could be an important vírus infecting AYB, resulting in significant yield reduction [3,13]. BCMV could pose a significant threat to AYB cultivation as it could be introduced into crops in new areas by distribution of infected seed [14]. There have been reports of significant yield loss to other legumes especially cowpea [15]. The identity of the virus infecting AYB was initially resolved using ELISA and confirmed using PCR followed by sequencing. The symptomatic leaves were positive using the BCMV and anti-potyvirus antisera, while there were pockets of mixed infectios with BCMV and CMV (3 plants) and were negative for the other antisera used (Data not shown). Five samples (Fig. 2) were positive out of seven tested, each giving the expected product size of 496 bp following PCR amplification.
The sequence from two isolates sequenced in this study were found to be identical and had the highest identities (99%) with those of BCMV isolate TN2 (KY044818) and TN-1 (KU761589) reported from infected cowpea (Vigna unguiculata) in India. Besides AYB isolates, the only other Nigerian BCMV isolate sequences that could be retrieved from sequence databases are isolates from Vigna unguculata [16].which has a 96% nucotide identity with AYB isolate from this study. The identities of the nucleotide and amino acid sequence of the CP gene of the virus isolated from this study relative to those of other BCMV were 82.96-99.1% and 87.33-100%, respectively (Table 1). Inoue-Nagata et al. [17] after examining several potyvirus sequences, suggested that the cutoff point for optimal CP nucleotide sequence recognition is < 76% and CP amino acid sequence recognition is < 82% in the same potyvirus species. In this study both the nucleotide and the amino acid sequences of the partial CP of the Nigeria AYB infecting virus vary in their identity by about 0.9-17 per cent with other BCMV isolates ( Table 1). In phylogenetic analyses based on the CPnucleotide sequences, isolates of BCMV were grouped into two major clusters (Fig. 3). Isolates BCMV-IART-AYB and BCMV-TN2 were in the same cluster with other BCMV strains of the peanut stripe (PSt) and the blackeye cowpea (BlC) strains. These results suggest that BCMV-IART-AYB is closely related to a BCMV strain Isolated from india. Therefore, more research into determination of the complete sequences of the virus is required as well as its impact on yield and other nutritional factors.