Molecular Characterization of Viruses Infecting Common Bean (Phaseolus Vulgaris L.) and Reaction of Bean Genotypes to Virus Infection

Abstract

Virus disease symptoms are frequent in common bean (Phaseolus vulgaris L.) fields in Zambia, but information is scanty about the identities, distribution and genetic diversities of causal pathogens. This study was conducted to survey for common bean virus-like diseases in farmers’ fields in Zambia, conduct molecular characterization of viruses identified and screen Zambian common bean cultivars for resistance to bean common mosaic necrosis virus (BCMNV) and bean common mosaic virus (BCMV). To address these knowledge gaps, surveys were conducted from March to May of 2018 in 128 common bean fields across six provinces of Zambia located in agro-ecological zones (AEZs) II and III. In total, 640 leaf tissue samples (symptomatic = 585; non-symptomatic = 55) were collected for virus diagnoses. From these, a subset of 223 samples that were selected based on symptom diversity and disease severity were subsumed into nine composite samples and subjected to total nucleic acid (TNA) extractions. Each of the nine TNA samples were diagnosed by high throughput sequencing (HTS) and the generated sequence reads were bioinformatically analyzed. Subsequently, the 640 samples were screened for the HTS-detected viruses using reverse transcription polymerase chain reaction (RT-PCR) and the results were validated by Sanger sequencing. Analysis of the combined HTS data obtained from composite samples produced nine distinct viruses belonging to five genera (Potyvirus, Cucumovirus, Endornavirus, Sobemovirus and Umbravirus). Screening of the 640 samples showed that 67% (429/640) of the samples were positive for at least one of the nine viruses either as single (65.1%; 417/640) or mixed (~1.9%; 12/640) infections. Southern bean mosaic virus (SBMV) was the most frequently detected virus accounting for ~29.4% (188/640) of the samples, followed by phaseolus vulgaris endornavirus1 (PvEV-1) ~9.2% (59/640). The remaining seven viruses, BCMV, BCMNV, cowpea aphidborne mosaic virus (CABMV), cucumber mosaic virus (CMV), Ethiopian tobacco bushy top virus (ETBTV), peanut mottle virus (PeMoV) and PvEV-2, occurred at incidences of 0.3% (2/640) for CABMV to 7.7% (49/640) for BCMNV and PvEV-2. Across the AEZ, there was more virus diversity in AEZ II (5 to 8 viruses) than in AEZ III (3 to 5 viruses). De novo assembly of the HTS generated sequence reads resulted in 24 virus-aligned sequences (BCMV=3, BCMNV=3, CABMV=2, ETBTV=2, PeMoV=2, CMV=1, PvEV-1=1, PvEV-2=1, SBMV=9). In pairwise comparison, ETBTV, SBMV and PeMoV sequences shared 88 to 99.4% nucleotide (nt) identities with corresponding sequences of respective global viruses whereas sequences of viruses CMV, BCMNV, BCMV, PvEV-1 and PvEV-2 shared 94 to 100% nt identities with corresponding global sequences. Further analyses revealed that the three BCMV sequences are putative recombinants whereas PeMoV isolate CP-com-1 and SBMV sequence Mse-3 are putative mutants. Phylogenetic analyses of the 24 virus sequences and global sequence homologs showed that virus sequences from this study clustered severally on the phylogenetic trees. For example, BCMNV and BCMV formed clusters with global isolates of known BCMNV and BCMV pathogroups (PGs). Thus, to establish PGs of BCMNV and BCMV from this study, the two viruses in the bean samples were assayed in standard differential common bean cultivars. Four PGs (I, III, VIa and VIb) were identified with the occurrence of PGs I and III in Zambia being reported for the first time in this study. The identified PGs were used to screen 14 common bean cultivars for resistance to BCMNV and BCMV. Two released varieties Lwangeni and Lunga that carry the resistance gene bc-3 were resistant to all four PGs whereas those bearing resistance gene bc-12 were susceptible to viruses in PGs VIa and VIb. Therefore, farmers are encouraged to plant the two varieties especially in BCMNV and BCMV hotspots. The results from this study will be used to design diagnostic tools for detecting vommon bean viruses in Zambia.