First Report of Ethiopian tobacco bushy top virus and Its Associated Satellite RNA Infecting Common Bean (Phaseolus vulgaris) in Zambia

During surveys for common bean (Phaseolus vulgaris L.) viruses in Central Province of Zambia in April 2018, symptoms of bushy top, deep green curled branches, and patchy leaf chlorosis were observed on five plants in a 2-ha farmer’s field. Total RNA was isolated from symptomatic leaf samples using the CTAB method (Chang et al. 1993). The RNA from one sample (CP414-1) was used to construct a cDNA library with the Illumina TruSeq RNA Library Prep Kit (Illumina, San Diego, CA), followed by high-throughput sequencing (HTS) on the Illumina MiSeq platform that generated ∼3.1 million single-end raw reads of ∼300 nucleotides (nt) each. A total of 355,885 reads showed hits to Ethiopian tobacco bushy top virus (ETBTV; Umbravirus), ETBTV satellite RNA (satRNA), and peanut mottle virus (PeMoV, Potyvirus) based on BLASTn analysis. The full-length genomes of ETBTV (4,239 nt; MT225089), its satRNA (521 nt; MT225092), and PeMoV (9,643 nt) were assembled from the HTS reads using Geneious R11.1.2 (Biomatters, Auckland, New Zealand). The obtained complete genome sequences of ETBTV (MT225089) and ETBTV satRNA (MT225092) shared 88 and 95% nt identities, respectively, with the corresponding viral (KJ918748) and satRNA (KJ918747) sequences of isolate 18-2 (Abraham et al. 2014). The near-complete PeMoV genome was 89% identical to isolate Liaoning (MH270528). The HTS results were validated by two-step reverse transcription polymerase chain reaction (RT-PCR) analyses of the five field-collected samples using newly designed primer pairs (data not shown). All five samples gave the expected 988-bp ETBTV-specific and 521-bp satRNA-specific DNA bands, and three samples produced the expected 2,100-bp PeMoV-specific fragment. The virus specificities of the agent-specific PCR fragments were ascertained by Sanger sequencing (ETBTV, MT225090 to 91; ETBTV satRNA, MT225093 to 94; PeMoV, MT900843 to 44), and they shared 98 to 100% nt identities with their corresponding HTS-derived sequences. 


Rabson Mulenga, Douglas Miano, Evans Kaimoyo, Juliet Akello, Felister Nzuve, Edgar Simulundu

Olufemi J. Alabi