Towards understanding the evolution of Banana bunchy top virus and the detection of associated badnaviruses

Abstract

Bananas are an important subsistence and export crop with over 130 tonnes of banana produced annually. Domesticated banana are triploid and sterile and it is thought that wild diploid bananas were domesticated between 7,000 and 10,000 years ago somewhere in the region of Indonesia, Philippines, New Guinea or the Southeast Asia peninsula. Triploid bananas were subsequently moved around the world through vegetative propagules and more recently through tissue culture.

A number of diseases are associated with bananas, including banana bunchy top disease (BBTD) and banana streak disease (BSD), both caused by DNA viruses. BBTD is caused by Banana bunchy top virus (BBTV), a single-stranded DNA (ssDNA) virus (genus Babuvirus; family Nanoviridae). Characteristic symptoms of BBTD are severe stunting of the plant and bunching of the leaves, with a green dot-dash streak on the underside of the leaves. Both viruses can cause severe crop loss and are of concern to banana growing regions. BSD is caused by a number of banana-infecting badnaviruses species (Badnavirus; Caulimoviridae), all of which contain a double-stranded DNA (dsDNA) genome. Characteristic symptoms of BSD include chlorotic and necrotic streaks on the leaves, which are not always seen across all leaves, or even across an entire leaf.

BBTV is a multi-component virus which is transmitted by the banana aphid Pentalonia nigronervosa. It is the type member of the babuviruses, which contains two other accepted species, Cardamom bushy dwarf virus (CBDV) and Abaca bunchy top virus (ABTV), which infect large cardamom and Musa spp, respectively. It is generally accepted that the genome of BBTV consists of six different components, with each component individually encapsidated. BBTV is able to evolve through recombination, reassortment and mutation. The large numbers of sequenced BBTV components in Chapter Two and Three (927), and publically available component sequences, have allowed for in-depth analyses into the diversity and evolution of this important plant pathogen. Our analysis shows that both reassortment and recombination play a significant role in the evolution of BBTV. Additionally, we found high genetic diversity in two geographic regions, the Southeast Asian / Far East region and the Indian subcontinent. Phylogenetic analysis of all full genomes, with recombinant regions and reassorted components removed, identified that the BBTV genomes circulating in the majority of countries have likely originated from single founder populations.

All nanoviruses contain two common regions which are common across species. The common region stem-loop (CR-SL) is involved in initiation of replication and is recognised by the replication-associated protein (Rep), encoded in the DNA-R component. The Rep recognises cognate components through iteron sequences in the CR-SL and nicks the component at the nonanucleotide motif to initiate replication. The common region major (CR-M) is involved in secondary strand replication. The large amount of sequence data generated in Chapters Two and Three, and a recent deposition of a large number of CBDV sequences, allowed a comprehensive analysis of the common regions of the babuviruses. All CR-SL across the three species showed high similarity including the iteron sequences. The CR-M regions however were not similar across the species. Alphasatellites, which only encode a Rep, have previously been found associated with some babuvirus, nanovirus and geminivirus isolates. Therefore these were also analysed for common regions. The babuvirus alphasatellites were more similar to the other alphasatellites than to the babuvirus components.

Both BBTV and banana-infecting badnaviruses are DNA viruses which infect the same host species, therefore the banana material that was collected for BBTV analyses was also screened for three species of banana-infecting badnaviruses. Species specific screening primers were designed for Banana streak MY virus (BSMYV), Banana streak OL virus (BSOLV) and Banana streak GF virus (BSGFV) across the movement protein motif located on open reading frame (ORF) three. With these primers we had a higher detection rate, for all three species, compared to previously published primers for banana-infecting badnaviruses. Eighty-two banana samples from 11 countries were found to be positive for at least one badnavirus, with the majority containing BSMYV. Of the 82 positive samples, 51 samples were also positive for BBTV. Banana-infecting badnaviruses are able to exist in two forms, a circular episomal infective form which is transmitted by a number of mealybug species and an endogenous form which is integrated into the banana genome and does not cause symptoms. However, a number of endogenous forms have been identified which are able to reactivate from the Musa genome into the episomal infectious form. As both forms are potentially able to cause infection, detection of either form is important in the identification of clean planting stock.

This PhD thesis investigated banana infecting DNA viruses, Banana bunchy top virus and three banana-infecting badnaviruses. In-depth analyses of the global diversity, evolution and the dispersal of BBTV were undertaken. The common regions of the babuviruses and alphasatellites were characterised and screening primers for BSMYV, BSOLV and BSGFV were designed.