Molecular characterization and diagnosis of badnaviruses infecting yams in the South Pacific by Amit Chand Sukal Bachelor of Science (Biology/Chemistry) Master of Science (Biology) Centre for Tropical Crops and Biocommodities School of Earth, Environment and Biological Sciences Faculty of Science and Technology A thesis submitted for the degree of Doctor of Philosophy Queensland University of Technology 2018 This page is intentionally left blank ii Abstract Yams (Dioscorea spp.) are economically important, annual or perennial tuber-bearing, tropical plants. Globally yam ranks as the fourth most important root crop by production and is a staple food crop for millions of people in Africa, the Caribbean, South America, Asia and the Pacific. In Pacific Island countries (PICs), the production and utilization of yams is limited by several factors including diseases and the lack of genetic diversity. An important global in vitro collection of yam germplasm is conserved in tissue culture by the Pacific Community’s (SPC) Centre for Pacific Crops and Trees (CePaCT) in Fiji. Evaluation of this germplasm and its distribution to PICs holds the key to improving production. However, similar to other vegetatively propagated crops, yam has a tendency to accumulate and perpetuate tuber-borne fungal and viral diseases. Although the tissue culture process eliminates fungal pathogens, viruses remain an issue. As such, quarantine regulations prohibit the movement of the yam germplasm from the SPC-CePaCT germplasm collection to other countries due to the risks associated with movement of untested and/or virus-infected material. To comply with these standards, sensitive diagnostic tests are needed to enable the virus indexing of yam germplasm. Several different viruses are known to infect yams, but badnaviruses, namely the Dioscorea bacilliform viruses (DBVs), remain the least studied and the most difficult to diagnose. The limited studies conducted on DBVs in PICs, using PCR-based studies, suggest that they are prevalent and are highly diverse. This high genetic variability hinders the development of reliable PCR-based diagnostic tests. DBV diagnostics is further complicated by the fact that badnavirus sequences are integrated in the genomes of some yam cultivars leading to false positives using PCR-based tests. Further, since all studies on DBV in the Pacific have been PCR- based, the existence of episomal DBV in Pacific yam remains unknown. Therefore, iii the aims of this PhD were to identify and characterize the diversity of episomal badnaviruses infecting yams in the Pacific to support the development of diagnostic protocols. A rolling circle amplification (RCA)-based approach, previously used for the characterization of episomal banana streak viruses (BSVs) from banana, was used in initial screening of yam accessions from SPC-CePaCT. Using RCA, two novel badnaviruses, namely Dioscorea bacilliform AL virus 2 (DBALV2) from Papua New Guinea (PNG) and Dioscorea bacilliform ES virus (DBESV) from Fiji were amplified and characterized. In addition, an isolate of Dioscorea bacilliform RT virus 2 (DBRTV2) was characterized from Samoa, which is the first report from the Pacific. Further, a novel viral sequence, tentatively named Dioscorea nummularia-associated virus (DNUaV), infecting D. nummularia from Samoa was identified. The genome size, organization, and the presence of conserved amino acid domains of DNUaV were found to be characteristic of members of the family Caulimoviridae. However, based on the criteria used for the demarcation of species in the family by the International Committee on Taxonomy of Viruses (ICTV), DNUaV is likely representative of a member of a new genus within the Caulimoviridae family. This was further supported by pairwise sequence analysis using pol gene sequences which showed 42 to 58% nucleotide and 27 to 53% amino acid identity between DNUaV and type members of other recognized genera within the family Caulimoviridae. Despite some success in using RCA for the characterization of DBVs from Pacific yams, in some cases the existing protocols yielded inconsistent results and produced background amplification of host circular DNA, such as plastids. Therefore, a suite of badnavirus-specific primers was designed from published sequences and used to optimize badnavirus-biased RCA protocols, such as directed-RCA (D-RCA) iv and specific primed-RCA (SP-RCA), using a commercially available phi29 polymerase. The optimized badnavirus RCA protocols performed up to 80-fold better than the commercially available TempliPhi kit-based random primed-RCA (RCA) based on Illumina MiSeq sequencing analysis. D-RCA was found to be the best protocol for badnavirus genome amplification and was subsequently used to test 224 yam accessions in the SPC-CePaCT Pacific yam germplasm collection including D. alata (185), D. esculenta (31), D. bulbifera (6), and one each of D. transversa and D. trifida. Thirty-five samples from three countries (PNG, Tonga and Vanuatu), representing five yam species (D. alata, D. bulbifera, D. esculenta, D. transversa and D. trifida) produced restriction profiles indicative of badnaviruses following digestion with EcoRI and SphI. Twenty samples were selected, and the SphI digested RCA products were cloned and sequenced using Sanger sequencing (n=4) or undigested RCA products sequenced using Illumina MiSeq (n=16) to obtain full length genome sequences. A total of 10 Dioscorea bacilliform AL virus (DBALV) genomes were generated from Vanuatu D. alata (n=2), D. bulbifera (n=3), D. esculenta (n=2), D. transversa (n=1) and D. trifida (n=1), or Tonga D. esculenta (n=1), while an additional 10 DBALV2 genomes were generated from PNG D. alata. This study also revealed that RCA, in combination with restriction analysis and/or Sanger sequencing and/or Next Generation Sequencing (NGS), could be successfully used for the detection and characterization of DBVs from Pacific yams. Such a strategy can now be used for the detection and further characterization of DBVs in the Pacific and other regions. An understanding of the episomal virus diversity infecting Pacific yam will help the further improvement of diagnostic protocols. This study has generated novel data that will support the global community in DBV diagnostics and also provides a foundation for the development of a consolidated v global diagnostic approach to enable the routine testing of yam germplasm. In the immediate future, the results of this study will enable the indexing of the yam collections, currently conserved at SPC-CePaCT, for DBVs and support the safe distribution and utilization of yam germplasm. Keywords: Dioscorea bacilliform virus (DBV), Dioscorea bacilliform AL virus (DBALV), Dioscorea bacilliform AL virus 2 (DBALV2), Dioscorea bacilliform ES virus (DBESV), Dioscorea bacilliform RT virus 2 (DBRTV2), rolling circle amplification (RCA), random-primed RCA (RP-RCA), directed RCA (D-RCA), specific-primed RCA (SP-RCA), next generation sequencing (NGS) vi Publications Peer reviewed publications related to this PhD thesis 1. Sukal, A., Kidanemariam, D., Dale, J., James, A. and Harding, R. (2017). Characterization of badnaviruses infecting Dioscorea spp. in the Pacific reveals two putative novel species and the first report of dioscorea bacilliform RT virus 2. Virus Research 238, 29–34. 2. Sukal, A., Kidanemariam, D., Dale, J., Harding, R. and James, A. (2018). Characterization of a novel member of the family Caulimoviridae infecting Dioscorea nummularia in the Pacific, which may represent a new genus of dsDNA plant viruses. PLos ONE 13, 1-12. 3. Sukal, A., Kidanemariam, D., Dale, J., Harding, R. and James, A. (2018). An improved degenerate-primed rolling circle amplification and next-generation sequencing approach for the detection and characterization of badnaviruses. Formatted for submission to Virology. 4. Sukal, A., Kidanemariam, D., Dale, J., Harding, R. and James, A. (2018). Characterization and genetic diversity of Dioscorea bacilliform viruses infecting Pacific yam germplasm collections. Formatted for submission to Plant Pathology. vii This page is intentionally left blank viii Table of Contents Abstract ............................................................................................................. ii Publications ................................................................................................... vii Table of Contents ........................................................................................... ix List of Figures ............................................................................................... xv List of Tables ............................................................................................... xvii List of Abbreviations.................................................................................... xix Statement of Original Authorship ............................................................. xxi Acknowledgements ..................................................................................... xxii Chapter 1 Introduction ........................................................................................................ 1 1.1 Description of scientific problem investigated ............................................ 1 1.2 Overall objectives of the study ..................................................................... 2 1.3 Specific aims of the study ............................................................................ 2 1.4 Account of scientific progress linking