Molecular identification and genetic variability analysis of babuviruses infecting banana (Musa spp.) and development of disease diagnostic protocol

Banana bunchy top disease (BBTD) caused by Banana bunchy top virus (BBT), genus Babuvirus, and family Nanovoridae, is a major constraint of the banana industry. In the Philippines, earlier studies on molecular characterization of BBTV infecting banana (Musa spp.) using virus isolates from two provinces (Negros and Laguna) have been undertaken. This study was conducted to determine the identity and genetic variability of virus isolates collected from 10 provinces across seven regions in the country and develop a molecular diagnostic protocol for virus detection. Analysis by enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) has detected the presence of BBTV in the virus isolates. On the other hand, the virus isolates tested negative for Abaca bunchy top virus (ABTV), a Babuvirus that also infects banana. Nucleotide BLAST analysis of the full-length DNA-R (rep) and DNA-S (coat protein) components identified BBTV in all the virus isolates analyzed in this study. All isolates shared the closest sequence identity (96.59- 99.59%) for DNA-R and DNA-S components with the BBTV reference sequences. Pairwise sequence identity of the local BBTV isolates ranged from 95.3-99.4% and 99.3- 98.2% in DNA-R and DNA-S, respectively. The BBTV isolates shared 96.9-99.6% identity with the Southeast Asia (SEA) and Pacific Indian Ocean (PIO) groups in DNA-R and DNA-S, indicating no distinct variation in the sequence. Phylogenetic analysis showed a high degree of homology of local virus isolates with SEA and PIO groups because they clustered in one major clade. Haplotype network grouping of virus isolates revealed three and four haplotype groups in DNA-R and DNA-S components, respectively. Local isolates showed very low nucleotide diversity in DNA-R (n=0119354) and DNA-S (n=0119354). Multiple sequence alignment showed few nucleotide variations throughout the length of the DNA. BBTV was detectable by PCR at different dilutions of Dellaporta-extracted DNA in fresh and dried leaf samples. In Alkali-extracted DNA, BBTV was detectable at different dilutions in fresh leaf samples, but in dried leaf samples, BBTV can be detected only at 10 sq and 10 cu dilutions. The knowledge of the identity and genetic variability of the babuvirus infecting banana is an important consideration for correct disease diagnosis and indexing of banana planting materials, as well as for BBTD surveillance and monitoring.