Upload
others
View
5
Download
0
Embed Size (px)
Citation preview
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
ii
This page is intentionally left blank
iii
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,
iv
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)
v
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
vi
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)
vii
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.
viii
This page is intentionally left blank
ix
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 the scientific papers ........................ 3
1.5 References .................................................................................................... 6
Chapter 2
Literature Review ................................................................................................ 7
2.1 Introduction .................................................................................................. 7
x
2.2 Taxonomy and morphology of yam ............................................................. 8
2.3 Origin and distribution of yam ................................................................... 11
2.4 Yam in the South Pacific ............................................................................ 12
2.5 Caulimoviridae ........................................................................................... 17
2.6 Badnaviruses .............................................................................................. 18
2.7 Detection of badnaviruses .......................................................................... 22
2.8 Current knowledge of badnaviruses infecting yam .................................... 27
2.9 Conservation and utilization of yams ......................................................... 30
2.10 Research problem and aim ......................................................................... 31
2.11 Objectives ................................................................................................... 31
2.12 References .................................................................................................. 32
Chapter 3
Characterization of badnaviruses infecting Dioscorea spp. in the Pacific reveals
two putative novel species and the first report of dioscorea bacilliform RT virus 2.
.......................................................................................................................... 43
Abstract .................................................................................................................. 45
Conflict of interest .................................................................................................. 60
Financial support .................................................................................................... 60
Acknowledgements ................................................................................................ 61
References .............................................................................................................. 61
Supplementary information .................................................................................... 66
xi
Chapter 4
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 ...................................................................................................... 69
Abstract .................................................................................................................. 70
Introduction ............................................................................................................ 71
Materials and methods ........................................................................................... 74
Plant material and nucleic acid extraction ......................................................... 74
RCA and sequencing .......................................................................................... 74
Sequence comparisons and phylogenetic analyses ............................................ 76
Virus detection ................................................................................................... 76
Results .................................................................................................................... 77
Identification of DNUaV ................................................................................... 77
Genome organization, sequence and phylogenetic analysis .............................. 78
PCR screening for DNUaV ................................................................................ 83
Discussion .............................................................................................................. 88
Acknowledgments .................................................................................................. 91
References .............................................................................................................. 92
Supporting Information .......................................................................................... 97
xii
Chapter 5
An improved degenerate-primed rolling circle amplification and next-generation
sequencing approach for the detection and characterization of badnaviruses .... 101
Abstract ................................................................................................................ 102
1. Introduction .................................................................................................. 103
2. Materials and Methods ................................................................................. 107
2.1. Samples ................................................................................................ 107
2.2. Primer design........................................................................................ 107
2.3. Random-primed RCA (RP-RCA) ........................................................ 108
2.4. Primer-spiked random-primed RCA (primer-spiked RP-RCA) ........... 109
2.5. Directed RCA (D-RCA) ....................................................................... 109
2.6. Specific-primed RCA (SP-RCA) ......................................................... 111
2.7. Optimization of D-RCA and SP-RCA ................................................. 111
2.8. Restriction analysis, cloning and Sanger sequencing........................... 111
3. Results .......................................................................................................... 113
3.1. Badnavirus RCA optimization ............................................................. 113
3.2. RP-RCA, primer-spiked RP-RCA, D-RCA and SP-RCA amplification
of badnaviruses ................................................................................................. 118
3.3. RCA-NGS for virus characterization ................................................... 122
3.4. DBALV isolate VUT02_De genome ................................................... 123
4. Discussion .................................................................................................... 124
Acknowledgements .............................................................................................. 127
References ............................................................................................................ 128
xiii
Chapter 6
Characterization and genetic diversity of Dioscorea bacilliform viruses infecting
Pacific yam germplasm collections ................................................................... 137
Abstract ................................................................................................................ 138
Introduction .......................................................................................................... 140
Methods ................................................................................................................ 141
Sample details, total nucleic acid (TNA) extractions....................................... 141
Viral DNA enrichment, RCA-RFLP, cloning and Sanger sequencing ............ 142
Next generation sequencing and genome assembly ......................................... 143
Pairwise sequence comparisons and phylogenetic analyses ............................ 145
Results .................................................................................................................. 145
RCA and Sanger sequencing ............................................................................ 145
Next generation sequencing of Pacific DBV isolates ...................................... 152
Analysis of DBALV and DBALV2 complete genomes .................................. 153
Dioscorea bacilliform AL virus (DBALV) from the Pacific ........................... 156
Dioscorea bacilliform AL virus 2 (DBALV2) from the Pacific ...................... 158
Discussion ............................................................................................................ 160
Acknowledgements .............................................................................................. 166
References ............................................................................................................ 167
Chapter 7
General Discussion .......................................................................................... 173
7.1. Dioscorea bacilliform virus (DBV) .......................................................... 175
xiv
7.2. Dioscorea nummularia-associated virus (DNUaV).................................. 176
7.3. Development of diagnostic protocols ....................................................... 178
7.4. Conclusions .............................................................................................. 181
7.5. References ................................................................................................ 184
xv
List of Figures
Chapter 2
Figure 1: FAO production data for yams from 2006-2016. ........................................ 9
Figure 2: Yam production issues in the Pacific. ....................................................... 15
Figure 3: Morphology of caulimoviridae particles ................................................... 19
Figure 4: Linear representation of a typical badnavirus genome organization ......... 21
Figure 5: The principle of rolling circle amplification ............................................. 25
Figure 6: Variable symptoms on yam leaves infected with badnaviruses. ............... 28
Chapter 3
Figure 1: Linearized representation of the genome organization of (A) DBESV
(isolate FJ14), (B) DBALV2 (isolate PNG10) and (C) DBRTV2-[4RT] . 52
Figure 2: Phylogenetic tree constructed using maximum likelihood method based on
the partial (A) RT/RNaseH-coding and (B) full length sequences of DBESV
(isolate FJ14), DBALV2 (isolate PNG10) and DBRTV2-[4RT] (isolate
SAM01) and previously described badnavirus sequences. ....................... 55
Chapter 4
Figure 1: Schematic representation of the genome organization of Dioscorea
nummularia-associated virus (DNUaV). ................................................... 80
Figure 2: Amino acid sequence alignments of the conserved motifs in the proteins of
the type member of each genus in the family Caulimoviridae. ................. 82
Figure 3: Phylogenetic analysis using the maximum-likelihood method following
ClustalW alignment in MEGA7 to infer evolutionary relationships of
DNUaV. ..................................................................................................... 86
xvi
Chapter 5
Figure 1: RCA of DBALV isolate VUT02_De to determine the effect of (A) gradient
incubation temperature, (B) dNTP concentration and (C) incubation
duration. ................................................................................................... 116
Figure 2: RCA of DBALV isolate VUT02_De using concentrations of 0 to 500 ng
total nucleic acid. ..................................................................................... 117
Figure 3: Different badnavirus infected samples amplified with (A) RP-RCA at 30°C
incubation, (B) RP-RCA at 36°C incubation, (C) primer-spiked RP-RCA at
30°C, (D) primer-spiked RP-RCA at 36°C, (E) D-RCA and (F) SP-RCA.
................................................................................................................. 121
Chapter 6
Figure 1: (A) EcoRI and (B) SphI restriction analysis of PNG D. alata RCA positive
samples. ................................................................................................... 148
Figure 2: (A) EcoRI and (B) SphI restriction analysis of Vanuatu (VUT) and Tonga
(TON) RCA positive samples. ................................................................. 149
Figure 3: PASC and phylogenetic analysis using the nucleotide of partial RT/RNase
H-coding nucleotide sequences showing the relationships of DBALV
isolates from this study with previously published complete DBALV
sequences.. ............................................................................................... 157
Figure 4: PASC and phylogenetic analysis using the nucleotide of partial RT/RNase
H-coding nucleotide sequences showing the relationships of DBALV2
isolates from this study with previously published complete DBALV2
sequences. ................................................................................................ 159
xvii
List of Tables
Chapter 2
Table 1: Genome characteristics of genera within the family Caulimoviridae......... 20
Chapter 3
Table S1: Arrangement of genome features of DBESV (isolate FJ14), DBALV2
(isolate PNG10) and DBRTV2-[4RT] (isolate SAM01). .......................... 66
Table S2: PCR primers used for the detection of DBESV (isolate FJ14), DBALV2
(isolate PNG10) and DBRTV2-[4RT] (isolate SAM01). .......................... 67
Chapter 4
Table 1: Mean pairwise nucleotide (above diagonal) and amino acid (below diagonal)
similarity between the pol gene of DNUaV and the type members of the
eight current genera within the family Caulimoviridae............................. 83
S1 Table: Details of yam partial RT/RNase H-coding sequences used in the
phylogenetic analysis of DNUaV. ............................................................. 97
S2 Table: Acronyms, GenBank accession and virus names of sequences used for
phylogenetic analysis in Fig 3B. ............................................................... 99
Chapter 5
Table 1: Sequences of primers used in primer-spiked RP-RCA, D-RCA and SP-RCA
protocols .................................................................................................. 110
xviii
Chapter 6
Table 1: Summary of badnavirus RCA testing results……………………….……146
Table 2: Arrangement of genomic feature of DBALV2 isolates obtained from
sequencing of RCA products ................................................................... 154
Table 3: Arrangement of genomic feature of DBALV isolates obtained from
sequencing of RCA products ................................................................... 155
xix
List of Abbreviations
aa amino acid
BLAST basic local alignment search tool
bp base pair/s
°C degrees Celsius
CePaCT Centre for Pacific Crops and Trees
CTAB cetyl trimethyl ammonium bromide
CTCB Centre for Tropical Crops and Biocommodities
DNA deoxyribonucleic acid
dNTP deoxynucleotide
ds double-stranded
DTT dithiothreitol
ELISA enzyme-linked immunosorbent assay
FAO Food and Agriculture Organization of the United Nations
IC-PCR immuno-capture polymerase chain reaction
ICTV International Committee on Taxonomy of Viruses
IR intergenic region
kbp kilobase pair/s
kDa kilodalton/s
mg milligram
min minute/s
µg microgram
µl microlitre/s
Mt million tonne/s
xx
NCBI National Centre for Biotechnology Information
NF nuclease free
ng nanogram/s
NGS next generation sequencing
nm nanometre/s
nt nucleotide/s
ORF open reading frame
PCR polymerase chain reaction
PICs Pacific Island Countries
QUT Queensland University of Technology
RCA rolling circle amplification
RFLP restriction fragment length polymorphism
ρmol picomole/s
RNA ribonucleic acid
RNase H ribonuclease H
RT reverse transcriptase
s second/s
SEF Science and Engineering Faculty
SPC Pacific Community
spp. species
U units
xxi
Statement of Original Authorship
The work compiled in this thesis has not been previously submitted to meet the
requirements of an award at this or any other higher education institution. To my
knowledge this thesis contains no previously published or written work of another
person except where due reference is made.
QUT Verified Signature
xxii
Acknowledgements
This project would not have eventuated if it were not for Grahame Jackson,
Michael Furlong and Richard Markham, and their passion to enhance food and
nutritional security through agricultural development in the Pacific. The importance
of yams for Pacific agriculture led to this project being funded through the Australian
Centre for International Agricultural Research (ACIAR) (#PC/2010/065). I also thank
ACIAR for the John Allwright Fellowship which provided funding for my PhD. I
would also like to acknowledge SPC-CePaCT for its continued efforts to support safe
conservation and utilization of plant genetic resource. SPC is a wonderful organization
which understands the need for staff capacity building and has supported this project
and made their yam collections available for this study.
A big thank you to my supervisors, Rob Harding, Anthony James and James
Dale, for their continued support, advice and supervision during my PhD. It was a
learning experience every time I had a chat with Rob and AJ, their guidance always
put me in the right direction. I will forever remember our accomplishments under this
project and our celebrations of our accomplishments. Ben, I believe a ‘Thank You
Mate’ is in order for all the well-placed advice, the rugby chats and beer shouts. The
CTCB crew thank you. My four years have gone by very quickly and I am thankful
for all the support, laughter, and the beers along the way. Dani your patience in dealing
with the lab is commendable. Thank you all for your help.
This acknowledgment would not be complete if I do not dedicate an entire
paragraph thanking my friend/brother, Dawit. You have been instrumental in teaching
me the tricks and trade of the pipette when I needed it the most. I would also like to
thank his better half Abigail, thanks for bearing with us when we left you and went out
for a few beers.
xxiii
This research would not have been successful without the support of my
family. I would like to thank my parents for their support, they are the reason I am
what I am today. The last couple of years have been hard for my family as we lost
something that was dear to our heart. Kapil my bro, you went doing what you loved,
we miss you and I dedicate this thesis in your name.
Above all I believe my wife, Anjani, should get my biggest thanks. You have
been a pillar of strength for me. Your encouragement and support have pushed me on
to reach this stage and I am grateful. Thanks for taking care of the fort while I have
been away. I would like to acknowledge my kids, Eesha and Darsh, sorry for being
away all this time but dad’s going to make up for the lost time. I love you guys.
1
Chapter 1
Introduction
This thesis is presented in ‘Thesis by Publication’ style containing a
comprehensive literature review section (Chapter 2) followed by four results chapters
(Chapters 3 to 6) and a general discussion chapter (Chapter 7). Chapter 3 has been
published in the journal Virus Research, Chapter 4 has been provisionally accepted for
publication in the journal PLoS ONE, while chapters 5 and 6 have been formatted for
submission to Virology and Plant Pathology, respectively. The presentation of each of
the results chapters follows the formatting style of the target journal.
1.1 Description of scientific problem investigated
Yam (Dioscorea spp.) is one of the major staple food crops of the Pacific Island
Countries, however, despite having high economic and cultural importance yam
production has declined. In the Pacific there is huge potential for development of yam
into a major export commodity and as a crop to support food security in the midst of
climate change. However, exploitation of yam to its full potential has been slow in
some of the Pacific Island Countries, mainly due to the lack of genetic diversity, which
prevents the selection of desirable agronomical traits such as pest and disease
resistance as well as those important for export markets, such as nutrition, quality, and
the shape and size of tubers. The improvement of yam is further hindered by the rare
and often male-dominated flowering of the Pacific yam (D. alata). Access to diversity
will enable the countries to screen/evaluate for the desired agronomical traits and
increase local production. The diversity to drive this exists as an in vitro collection
conserved with the SPC-CePaCT. This unique collection has been amassed from the
2
Pacific and has the potential to support selection for the desired crop traits. However,
utilization of this unique collection has been very limited. The main reason for limited
utilization of the collection is due to the unavailability of diagnostic protocols to test
the collections for viruses, mainly the badnavirus -Dioscorea bacilliform virus (DBV).
To address this knowledge gap, the current PhD study was commissioned. The study
was aimed at characterizing the diversity of DBVs prevalent in the Pacific and
developing protocols for DBV indexing. Ultimately, the results of this study are aimed
at supporting the indexing of yams conserved with SPC-CePaCT to enable access to
genetic diversity by the PICs to increase utilization for enhanced food and nutrition
security in the Pacific.
1.2 Overall objectives of the study
The overall objective of this study was to characterize the diversity of episomal
Dioscorea bacilliform viruses present in Pacific yam and develop diagnostic protocols
to support safe exchange of yam germplasm.
1.3 Specific aims of the study
The specific aims of this project were to (i) screen the pacific germplasm collections
conserved at SPC-CePaCT for episomal DBV sequences, (ii) characterize the
molecular diversity of the episomal DBV sequences existent in the Pacific and (iii)
develop diagnostic protocols for the detection of DBVs.
3
1.4 Account of scientific progress linking the scientific papers
The SPC-CePaCT yam collection comprising of D. alata, D. rotundata, D.
esculenta, D. bulbifera, D. nummularia, D. transversa and D. trifida originating from
different Pacific Island Countries, namely Fiji, Federated States of Micronesia (FSM),
New Caledonia, Papua New Guinea (PNG), Vanuatu, Samoa and Tonga, was
acclimatized in the SPC-CePaCT screenhouse for at least three months. Total nucleic
acid (TNA) were extracted and brought to QUT for DBV screening. Previously
described RCA protocol (James et al., 2011) and restriction analysis using a number
of endonucleases (EcoRI, BamHI, KpnI, SalI and StuI), determined from published
badnavirus sequences and from experimentation, were used to screen samples for
potential badnavirus (DBV) sequences. Following RCA and restriction analysis, one
sample from Fiji, two samples from PNG and one sample from Samoa produced
restriction profiles indicative of DBV. The RCA restriction fragments were sequenced
and identified as three distinct species, Dioscorea bacilliform AL virus 2 (DBALV2),
Dioscorea bacilliform ES virus (DBESV) and Dioscorea bacilliform RT virus 2
(DBRTV2) from the PNG, Fiji and Samoa samples, respectively. At the time of
identification all three were novel species, however, during the course of manuscript
preparation, DBRTV2 was published by Bömer et al. (2016). Since there was no
previous sequence information for DBALV2 and DBESV, and the Pacific isolate of
DBRTV2, the complete genome sequences of each of the species was generated and
the results are presented in Chapter 3.
During further RCA screening and sequencing an additional previously
unidentified sequence group was detected from D. nummularia originating from
Samoa. The polymerase (pol) gene sequence was determined and compared to
published DBV and badnavirus sequences. Based on pol gene nucleotide sequence
4
comparisons, it was found that the amplified sequence had very low nucleotide
sequence identity (42-58%) with published badnavirus sequences. The RCA
restriction fragments were cloned and sequenced to generate the complete genome
sequence. The complete genome was found to be typical of a member of the family
Caulimoviridae but was different from the members of other genera in the family.
During phylogenetic studies it clustered between genus Badnavirus and genus
Tungroviruses, however, it had very low sequence identity with members of both
genera. It was determined to represent a new member of the family Caulimoviridae
and possibly a new genus within the family. Chapter 4 discusses in detail the new
sequence group identified, which is tentatively named as Dioscorea nummularia-
associated virus (DNUaV), giving evidence for the sequence to be considered
representative of a new genus within the family Caulimoviridae.
Following initial RCA screening using the RCA kits with added primers as
described previously (James et al., 2011) it was found that the protocol was
inconsistent and non-reproducible for DBV amplification from yam. Where
amplification was achieved, it was also found that a lot of the amplified products were
of plant origin, such as plastid. Eventually, the RCA protocol as used by James et al
(2011) was determined to be unsuitable for DBV amplification, as also suggested by
other authors (Bömer et al., 2016; Umber et al., 2014). RCA for badnavirus
amplification was improved by manipulating the individual components of the RCA
using the Phi29 enzyme (ThermoFisher Scientific) as previously described for low-
copy number human papillomavirus amplification (Marincevic-zuniga et al., 2012;
Rockett et al., 2015). Using 182 badnavirus complete genome sequences, representing
43 species, 28 primers were designed and together with the BadnaFP/RP primers
(Yang et al., 2003) and Badna-MFP/MRP primers (Turaki, 2014) were optimized for
5
use in two RCA protocols, namely directed RCA (D-RCA) and specific-primed RCA
(SP-RCA). Chapter 5 describes the optimization of the different D-RCA and SP-RCA,
with comparisons to previously described RCA protocols. D-RCA and SP-RCA were
both found to reliably amplify badnavirus DNA, however, D-RCA was found to be
the most efficient method. Subsequently, D-RCA followed by restriction analysis
and/or Sanger and/or next generation sequencing (NGS) was used to screen the entire
Pacific collection and characterize the episomal DBV diversity prevalent in Pacific
yams. The findings are summarised in Chapter 6.
This study is the first comprehensive survey of episomal DBV present in
Pacific yams. Two novel DBV species (DBALV2 and DBESV), two previously
characterized DBV species (DBALV and DBRTV2), and a novel virus genome which
may represent a new genus in the family Caulimoviridae have been described in this
study. The sequence information generated has been deposited in the National Centre
for Biotechnology Information (NCBI) GenBank database and will be available to the
global community to further the global initiatives on safe yam germplasm exchange.
Further, the described RCA and PCR-based protocols can be used for testing DBALV,
DBALV2, DBESV, DBRTV2 and DNUaV and expanded further to cover a wider
range of DBVs.
6
1.5 References
Bömer, M., Turaki, A., Silva, G., Kumar, P., Seal, S., 2016. A sequence-independent
strategy for amplification and characterization of episomal badnavirus sequences
reveals three previously uncharacterized yam badnaviruses. Viruses 8, 188.
James, A.P., Geijskes, R.J., Dale, J.L., A., Harding, R.M., 2011. Development of a
novel rolling-circle amplification technique to detect banana streak virus that also
discriminates between integrated and episomal virus sequences. Plant Dis. 95,
57–62.
Marincevic-Zuniga, Y., Gustavsson, I., Gyllensten, U., 2012. Multiply-primed rolling
circle amplification of human papillomavirus using sequence-specific primers.
Virology 432, 57-62.
Rockett, R., Barraclough, K.A., Isbel, N.M., Dudley, K.J., Nissen, M.D., Sloots, T.P.,
Bialasiewicz, S., 2015. Specific rolling circle amplification of low-copy human
polyomaviruses BKV, HPyV6, HPyV7, TSPyV, and STLPyV. J. Virol. Methods
215–216, 17–21.
Turaki, A.A., 2014. Characterization of badnavirus sequences in West African Yams
(Dioscorea spp.). PhD thesis, University of Greenwich, United Kingdom, 240.
Umber, M., Filloux, D., Muller, E., Laboureau, N., Galzi, S., Roumagnac, P., Iskra-
Caruana, M.-L., Pavis, C., Teycheney, P.-Y., Seal, S.E., 2014. The genome of
African yam (Dioscorea cayenensis-rotundata complex) hosts endogenous
sequences from four distinct badnavirus species. Mol. Plant Pathol. 15, 790–801.
Yang, I.C., Hafner, G.J., Dale, J.L., Harding, R.M., 2003. Genomic characterization
of taro bacilliform virus. Arch. Virol. 148, 937–949.
7
Chapter 2
Literature Review
2.1 Introduction
Yams (Dioscorea spp.) are classified in the family Dioscoreaceae and are
represented by some 644 species (Lebot, 2009). Ten of these species (D. alata, D.
cayenensis, D. nummularia, D. opposita, D. rotundata and D. transversa, D.
esculenta, D. bulbifera, D. trifida and D. pentaphylla) are economically important as
cultivated crops. Cultivated yam is ranked as the fourth most important root crop by
production after potato, sweet potato and cassava. In 2016, global production of yam
was estimated at around 66 million tonnes (Mt) with production in the Pacific region
being less than half a million ton (FAOSTAT, 2018). Cultivated yam provides a staple
food for millions of people in Africa, South America, Asia and the Pacific, while wild
yam provides a valuable source of food in times of famine and scarcity (Risimeri,
2001). They also provide valuable pharmacologically active compounds in traditional
medicine (Lebot, 2009). Yam production is highest in West Africa, which accounts
for 95% of the world’s total production (Asiedu and Sartie, 2010; FAOSTAT, 2018,
Mignouna et al., 2008). Dioscorea cayenensis-rotundata is by far the most common
species complex cultivated in this region, however, D. alata and D. esculenta are
predominant in Pacific Island Countries (PICs) (Kenyon et al., 2008; Lebot, 2009).
Apart from being a food staple, yam tubers are of great ceremonial significance in
many PICs, such as Papua New Guinea, Vanuatu, Fiji, Tonga and Pohnpei (Elevitch
and Love, 2011). Yam has the potential to become an important export commodity for
some of the PICs, especially to niche markets in countries where Pacific Islanders have
settled, such as Australia, New Zealand and the United States (SPYN, 2003; Sukal,
8
2015). Despite its importance, production of yams in the PICs has not increased to
meet the demand. FAO data for yam production in the PICs shows a mere 88 thousand
tonnes increase over the ten years from 2006 to 2016 compared to a global production
increase of 13 million tonnes for the same period (Figure 1). However, the relative
increase in the Pacific is about the same as that globally. This increase in PICs yam
production is far less than the demand that exists. Unavailability of suitable planting
material for desired agronomical traits as well as pests and diseases, such as
anthracnose disease caused by the fungus Colletotrichum gloeosporioides, are some
of the major constraints to production.
2.2 Taxonomy and morphology of yam
Dioscorea spp. are annual or perennial tuber-bearing, dioecious, climbing,
tropical monocots comprising the largest genus within the family Dioscoreaceae and
consisting of about 644 species (Govaerts et al., 2007; Lebot, 2009; Mignouna et al.,
2007). The genus Dioscorea is further divided into taxonomic sections - the 10 species
that are used as food crops belong to five different sections, namely Enantiophyllum
(D. alata, D. cayenensis, D. nummularia, D. opposita, D. rotundata and D.
transversa), Combilium (D. esculenta), Opsophyton (D. bulbifera), Macrogynodium
(D. trifida) and Lasiophyton (D. pentaphylla) (Lebot, 2009). Both annual and
perennial types occur, with the roots and stems renewing annually after spending the
dry part of the year in dormancy, a period which can vary from one to six months.
9
Figure 1: FAO production data for yams from 2006-2016. (A) World yam production
data; and (B) PICs yam production data (FAOSTAT, 2018).
40
45
50
55
60
65
70
Mil
lio
n t
on
nes
A
320
340
360
380
400
420
440
Th
ou
san
d t
on
nes
B
10
Tuber morphology, stem twining direction, dioecy and fruit/seed wing shape
are the most important phenotypic characters for the systematic classification of the
genus (Wilkin et al., 2005). Yams in the Enanthiophyllum section produce one to three
large tubers and have winged stems twining to the right (anticlockwise), with
occasional bulbils. Similarly, yams in the Combilium section have stems that also
twine right (anticlockwise) but have numerus smaller tubers. Yams of the Opsophyton
section produce aerial bulbils with the stems twining to the left (clockwise), while
Macrogynodium produce small tubers, with spineless stems twining to the left
(clockwise). Lasiophyton section produces a cluster of medium-sized tubers, with
stems twining to the left (clockwise) and with large thorns on the stem (Lebot, 2009).
However, there still remains some controversies regarding yam taxonomy, particularly
for the cultivated guinea yams and their wild relatives (Girma et al., 2016; Mignouna
et al., 2002; Ramser et al., 1997; Terauchi et al., 1992).
Cytological studies on Dioscorea spp. initially determined the basic
chromosome number of x=9 and x=10 in species from America, Europe and Africa,
while species in Asia and Oceania all have the basic chromosome number of x=10
(Essad, 1984). However, further studies have revealed that D. rotundata, which was
previously thought to be a tetraploid species (2n=40), is a diploid with a basic
chromosome number of x=20 (Scarcelli et al., 2005). Another study notes that D.
trifida (2n=80), which was considered to be octoploid, is actually a tetraploid with a
basic chromosome number of x=20 (Bousalem et al., 2006). Dioscorea alata was
considered to have a basic chromosome number of x=10 by many authors (Abraham
and Nair, 1991; Gamiette et al., 1999; Malapa et al., 2005), however, a more recent
study provided genetic evidence to confirm diploidy of plants with 2n=40
chromosomes, hence, supporting the hypothesis that plants with 2n=40, 60 and 80
11
chromosomes are diploids, triploids and tetraploids, respectively, with the basic
chromosome number of D. alata being x=20 (Arnau et al., 2009). Furthermore,
variable ploidy levels have also been observed within D. nummularia (2n=3x=60 to
2n=6x=120) (Lebot et al., 2017).
2.3 Origin and distribution of yam
The major cultivated yam species are believed to have originated in the tropical
areas of three separate continents, including (i) Africa (mainly West Africa for D.
rotundata, D. cayenensis and D. dumetorum), (ii) the region comprising South-East
Asia and the South Pacific (D. alata and D. esculenta) and (iii) South America (D.
trifida) (Arnau et al., 2010; Ayensu and Coursey, 1972; Bhattacharjee et al., 2011).
The occurrence of Dioscorea spp. in southern Asia, Africa and South America
predates human history, with domestication events occurring independently in
America, Africa, Madagascar, South and South-East Asia, and Oceania (Arnau et al.,
2010; Ayensu and Coursey, 1972; Bhattacharjee et al., 2011; Lebot, 2009). Although
debate still exists on the origin of D. alata, as it has not yet been found in its wild state
in nature, studies using AFLP markers show that it is closely related to D. nummularia
and D. transversa (Malapa et al., 2005), which are restricted to the South-East Asian
islands and Oceania. Therefore, by association, it has been proposed that D. alata may
belong to a South Asian-Oceanic gene pool that is confined to the former Sahulian and
Wallacean regions (Arnau et al., 2010; Lebot, 1997).
Yam cultivation occurs in many tropical regions. Based on annual production
data published by the Food and Agriculture Organization for the United Nations
(FAO) statistics, yam is cultivated in 61 different countries (FAOSTAT, 2018).
However, this is not a comprehensive list as some countries (such as China) do not
12
provide annual production statistics to FAO. D. alata is the most widely distributed
species in the humid and semi-humid tropics and, together with D. rotundata and D.
cayenensis which are indigenous to West Africa, is the most important yam in terms
of quantity produced and marketed (Asiedu and Sartie, 2010; Lebot, 2009). D. alata
(also referred to as greater yam) together with D. esculenta (also referred to as lesser
yam) is the most important cultivated yam species in the Pacific. Although, D.
esculenta is important as a staple food species and is the dominant species grown by
yam-dependent communities of Papua New Guinea, D. alata still retains a high status
from its use in cultural and ceremonial purposes throughout the Pacific (O’Sullivan,
2010).
2.4 Yam in the South Pacific
Yam is one of the most important food staples of the Pacific. It ranks among the
top crops in the tropics along with cassava (Manihot esculenta) and aroids (Colocasia
spp. and Xanthosoma spp.) (Elevitch and Love, 2011). It is also considered a
traditional crop with great ceremonial significance in many areas of the Pacific, such
as Papua New Guinea, Vanuatu, Fiji, Tonga and Pohnpei, and its cultivation is
consistent with maintaining a fragile ecosystem, such as those of the lowland areas,
where they are typically cultivated (Elevitch and Love, 2011; SPYN, 2003). Across
the Pacific region, yam cultivation is largely seasonal with the dominant cultivated
species being the greater yam (D. alata) and lesser or sweet yam (D. esculenta).
However, there is scattered cultivation of D. rotundata, D. bulbifera, D. nummularia,
D. transversa and D. trifida throughout the region.
Yam is considered as one of the crops with good potential for commercial
exploitation in the Pacific as niche markets become available in Australia, New
13
Zealand and the United States through Pacific Islander migration (SPYN, 2003; Sukal
et al., 2015). However, there are several potential issues which may hinder commercial
exploitation. For example, cultivation is very resource intensive and requires costly
materials that are in short supply, such as for staking (Figure 2A). Further, harvesting
of some cultivars due to tuber size is time-consuming and laborious compared to other
staple food crops in the Pacific (Figure 2B). The lack of information on the nutritional
content of the different yam species and cultivars hinders potential utilization of yam
as a high-quality vegetable (Figure 2C). The rare and often male-dominated flowering
of D. alata also prevents crop improvement for agronomic traits as well as for
resistance to pests and diseases, such as anthracnose (Figure 2D), which decreases the
production of many cultivars in the Pacific (SPYN, 2003; Sukal et al., 2015).
Therefore, there is an urgent need to collate and evaluate existing yam genetic
resources present in the Pacific to select for desired traits. Through the efforts of the
Pacific Community (SPC) under a European Union (EU)-funded South Pacific Yam
Network (SPYN) project, yam genetic resources (mainly D. alata) from the Pacific
have been collected and characterized. This clonal selection was subsequently
conserved at the Centre for Pacific Crops and Trees (CePaCT), the genebank of SPC
located in Suva, Fiji, in addition to in situ collections. This yam collection continues
to expand as new species and varieties are received from within and outside of the
Pacific region. Despite its importance, most of the collection has remained unavailable
for distribution, with only 10% being distributed throughout the region. This has been
primarily due to the threat of spreading diseases in the vegetative plant material.
14
A
B
D
15
C
D
Figure 2: Yam production issues in the Pacific. (A) Typical staking-type production
system in Fiji; (B) and (C) depict the varied size and shapes and colours of tubers from
D. alata, (with the purple tubers having high anthocyanin content); and (D)
anthracnose disease damage on a Fijian yam cultivar ‘Taniela’ of D. alata.
16
Like other vegetatively propagated crops, yam has a tendency to accumulate
and perpetuate tuber-borne fungal and viral diseases (Kenyon et al., 2008). Tissue
culture, which is the preferred method of yam germplasm exchange, helps eliminate
fungal pathogens, however, viruses remain an issue. Viruses belonging to the families
Alphaflexiviridae (genus Potexvirus), Betaflexiviridae (genus Carlavirus),
Bromoviridae (genus Cucumovirus), Caulimoviridae (genus Badnavirus), Potyviridae
(genus Macluravirus and Potyvirus), Secoviridae (genus Comovirus and Fabavirus)
and Tombusviridae (genus Aureusvirus) are known to infect yams (Kenyon et al.,
2001; Menzel et al., 2014).
Yam production is reduced by virus infection in all yam-growing areas (Fuji et
al., 1999; Kenyon et al., 2008; Mantell and Haque, 1978). Viruses also prevent/limit
the exchange of yam germplasm due to the risks associated with movement of untested
and/or virus-infected material. Quarantine regulations restrict the exchange of planting
material between PICs and West Africa unless plant material is certified as disease-
free. Even germplasm movement between the PICs is prohibited by in-country
quarantine regulations under the minimum phytosanitary standards set out by
quarantine legislation. To comply with these standards, sensitive diagnostic tests are
needed to enable the virus indexing of yam germplasm (Kenyon et al., 2008).
Of the nine different virus genera known to infect yams, the badnaviruses
remain the least studied and the most difficult to diagnose. Studies on badnaviruses
infecting yam suggest that they are highly diverse and have a high prevalence in Pacific
yams (Kenyon et al., 2008) which complicates detection efforts. The difficulty in
detection is further complicated by the fact that partial badnavirus sequences have been
found to integrate into the yam genome (Bousalem et al., 2009; Kenyon et al., 2008;
Seal et al., 2014; Umber et al., 2014). The results from previous studies also suggests
17
that the detection of badnaviruses in yam remains unreliable due to the high sequence
diversity of field isolates (Bömer et al., 2016; Bousalem et al., 2009; Kenyon et al.,
2008; Seal et al., 2014; Umber et al., 2014).
2.5 Caulimoviridae
Badnaviruses are one of eight genera within the family Caulimoviridae. All
members consist of reverse-transcribing, double-stranded deoxyribonucleic acid
(dsDNA) containing plant viruses, which are distinguished from each other primarily
by genome organization (Geering, 2014; Geering and Hull, 2012). Six of the genera,
Caulimovirus, Cavemovirus, Petuvirus, Rosadnavirus, Soymovirus and Solendovirus
have isometric virions that are 52 nm in diameter with an icosahedral T=7 symmetry,
while two genera, Badnaviruses and Tungroviruses, have bacilliform virions, with
dimensions of 30 x 130-150 nm and are tubular structures based on a T=3 icosahedron
cut across its threefold axis (Geering, 2014; Hull, 1996) (Figure 3). The genome size
of the circular dsDNA is between 7.2 and 9.2 kbp (Table 1) with all the coding capacity
on the positive-strand. The different genera are distinguished by the organization of
their open reading frames (ORFs) and there can be between one and seven ORFs
depending on the genus (Geering, 2014) (Table 1).
For some viruses within the genera Badnavirus, Petuvirus, Solendovirus and
Caulimovirus, viral DNA has been shown to be integrated within the host nuclear
genome. This integration of viral sequences into the host genome is referred to as
endogenous viral elements (EVEs) and is the result of illegitimate recombination, with
the integrated sequences being fragmented and rearranged when compared to the
respective ancestral virus genomes (Geering, 2014). EVEs are not restricted to the
family Caulimoviridae, but have also been reported from the Geminiviridae family
18
(Ashby et al., 1997; Bejarano et al., 1996), from host plants such as Nicotiana spp.,
and Dioscorea spp. (Filloux et al., 2015). In contrast, a greater diversity of EVEs
belonging to the four genera of Caulimoviridae (referred to as endogenous
pararetroviruses (EPRVs)) have been characterized in several plant species such as
tobacco, banana, bitter orange, fig, petunia, rice, potato and relatives, lucky bamboo,
tomato, Dahlia, pineapple, grapes, poplar and yam (Chabannes and Iskra-Caruana,
2013; Geering et al., 2010; Mette et al., 2002; Staginnus et al., 2009; Staginnus and
Richert-Pöggeler, 2006; Umber et al., 2014).
2.6 Badnavirus
Badnaviruses typically contain three open reading frames (ORFs) with ORF 1
encoding a protein of unknown function, ORF 2 coding for a virion-associated protein
(VAP) and ORF 3 coding for a large polyprotein which is processed into several
mature proteins including a movement protein (MP), coat protein (CP), an aspartic
protease (AP) reverse transcriptase (RT) and ribonuclease H (RNase H) (Geering,
2014; Olszewski and Lockhart, 2011) (Figure 4). However, Sweet potato pakakuy
virus (SPPV), which has a typical badnavirus genome organization, has the ORF 3
divided into two, namely ORF 3a (with the MP and CP domains) and ORF 3b (with
AP, RT and RNase H) (Geering, 2014; Kreuze et al., 2009).
Transmission of badnaviruses is mainly by vegetative propagation, mealybugs
and through seeds (Bhat et al., 2014; Huang and Hartung, 2001; Lockhart et al., 1997;
Yang et al., 2003a, 2003b). However, transmission of Rubus yellow net virus (RYNV),
Gooseberry vein banding associated virus (GVBAV) and Spiraea yellow leaf spot
virus (SYLSV) are by aphid vectors, while Piper yellow mottle virus (PYMoV) is
19
transmitted by both the citrus mealybug (Planococcus citri) and the black pepper
lacebug (Diconocoris distanti) (Geering, 2014).
Figure 3: Morphology of caulimoviridae particles. (Top left) reconstruction of the
surface structure of a cauliflower mosaic virus particle showing T=7 symmetry. (Top
right) Cutaway surface reconstruction showing multilayer structure (Adapted from
Cheng et al., 1992). (Bottom) Negative contrast electron micrograph of particles of
Commelina yellow mottle virus, stained with 2% sodium phosphotungstate, pH 7.0
(bar represents 10 nm) (Adapted from Geering and Hull, 2012).
20
Table 1: Genome characteristics of genera within the family Caulimoviridae (Geering,
2014).
Genus Genome size (kbp) No. of open reading frames (ORFs)
Badnavirus 7.2-9.2 3-4
Caulimovirus 7.8-8.2 6
Cavemovirus 7.7-8.2 4
Petuvirus 7.2 1
Rosadnavirus 9.3 8
Solendovirus 7.8-8.8 4
Soymovirus 8.1-8.2 7
Tungrovirus 8.0 4
21
Figure 4: Linear representation of a typical badnavirus genome organization showing the positions of the putative tRNAmet-binding site (tRNAmet),
TATA box and polyadenylation signal (polyA); open reading frames ORF 1; ORF 2; ORF 3 showing movement protein (MP), capsid protein (CP),
zinc finger (Zn), aspartic protease (AP), reverse transcriptase (RT) and ribonuclease H (RNase H) motifs.
22
2.7 Detection of badnaviruses
Detection of badnaviruses is complicated by their high serological and genetic
heterogeneity (Harper et al., 2005; Kenyon et al., 2008; Lockhart, 1986; Muller et al.,
2011; Seal et al., 2014). Polyclonal antibodies have been prepared against a cocktail
of approximately 30 purified isolates of banana streak virus (BSV) and sugarcane
bacilliform virus (SCBV). Although the antisera (referred to as BenL antisera) has
been raised against BSV and SCBV isolates, this polyclonal antibody mixture appears
to cross-react with other badnavirus species. It has been used for badnavirus detection
from crops such as bananas, yams and others using enzyme-linked immunosorbent
assay (ELISA) and immunosorbent electron microscopy ISEM (Le Provost et al.,
2006; Seal et al., 2014). However, the heterogeneity of badnaviruses in these crops, as
well as the fact that badnaviruses are often present at a low titre in infected plants,
means that not all infections are detected (Phillips et al., 1999; Seal et al., 2014; Yang
et al., 2003b), which renders these techniques unsuitable for routine diagnostics. Seal
et al. (2014) showed that although the BenL antisera detected some badnavirus isolates
from yams it failed to detect all isolates. Therefore, nucleic acid-based techniques, due
to their increased sensitivity, have been preferred for the detection of badnaviruses.
Based on the consensus sequence of the RT/RNase H-coding region of published
badnavirus sequences, a pair of degenerate primers (BadnaFP/RP) were designed
(Yang et al., 2003b) and have been used widely for badnavirus detection in a range of
host plants, including taro, yam, and sugarcane (Bousalem et al., 2009; Guimarães et
al., 2015; Kenyon et al., 2008; Seal and Muller, 2007; Yang et al., 2003a, 2003b). The
sequence of the core RT/RNase H-coding region amplified using these primers is not
only important for identification of badnaviruses but is also used for taxonomic
classification within the genus (Geering and Hull, 2012). According to the
23
International Committee on Taxonomy of Viruses (ICTV), a threshold of 20%
dissimilarity in nucleotide identity in this region is used as the criterion for the
demarcation of a new species. However, the discovery of integrated badnavirus
sequences in plant host genomes, as reported in banana (Harper et al., 1999; Le Provost
et al., 2006; Ndowora et al., 1999) and yam (Dioscorea cayenensis-rotundata) (Umber
et al., 2014), complicates the use of these primers for diagnosis. Although the
BadnaFP/RP primers have been shown to successfully detect many badnaviruses, not
all amplification generated is from episomal viral sequences since integrated
sequences can also act as a template in PCR (Seal et al., 2014). Immuno-capture PCR
(IC-PCR), which combines serological and molecular approaches, is another
diagnostic method used for detection of badnaviruses. However, IC-PCR relies on
antisera successfully trapping virus particles as well as the complete elimination of
genomic DNA from the reaction tubes, which is often difficult. IC-PCR using the
BenL antisera has been used successfully to detect badnaviruses in bananas (Le
Provost et al., 2006), however, these authors showed that genomic DNA can bind to
the tube and amplification can also result from integrated BSV sequences using IC-
PCR.
Rolling circle amplification (RCA) is a more recent method that has shown
promise in the detection of badnaviruses. RCA is a simple and efficient isothermal
enzymatic process that utilizes unique DNA (Phi29, Bst, and Vent exo-DNA
polymerase) or RNA (T7 RNA polymerase) polymerases to generate tandem repeats
of single-stranded circular DNA and RNA templates. During RCA, the polymerase
continuously adds nucleotides to a primer annealed to the template and, since it can be
isothermal, there is no need for a thermal cycler or a thermostable polymerase (Ali et
al., 2014). Among the different polymerases available for RCA, bacteriophage phi29
24
DNA polymerase has shown great potential for the amplification of viral circular
DNA. Phi29 possesses several features, such as strand displacement activity, proof-
reading activity and generation of very long synthesis product (Figure 5A-C), that
make it ideal for the efficient amplification of circular DNA molecules from complex
biological samples (Johne et al., 2009). These properties of phi29 DNA polymerase
are useful in the study of small circular DNA molecules and phi29 polymerase-
dependent RCA has been used in the study of several virus families with circular DNA
molecules (James et al., 2011a; Johne et al., 2009). The ability of phi29 to support
sequence independent amplification of circular DNA molecules, as long as a primer
binds to the template, means specific primers are not required and the RCA can be
carried out with random primers. This characteristic of phi29 polymerase has led to
the discovery of many novel DNA viruses infecting humans, animals and plants (Johne
et al., 2009). As observed in Figure 5C, multiprimed-RCA (using either specific or
random primers) leads to the production of long concatemeric molecule(s) of double-
stranded DNA. The concatemer can be subjected to restriction fragment length
polymorphism (RFLP) analysis. Digestion with a single cutting endonuclease can
produce a full-length monomeric product that can subsequently be purified, cloned and
sequenced.
25
Figure 5: The principle of rolling circle amplification (adapted from Johne et al.,
2009). Blue lines denote target DNA sequences, green lines represent oligonucleotide
primers and red lines represent new DNA synthesized by the polymerase. Arrowheads
indicate 3` ends of the synthesized DNA strands. (A) Linear template and single
primer. After primer binding, the polymerase synthesizes one complementary strand;
(B) circular template and single primer. The polymerase synthesizes a complementary
strand beginning at the bound primer. After one round, the primer and the synthesized
strand are displaced, and DNA synthesis continues for additional rounds. By this, a
long concatemeric single-stranded DNA is produced; and (C) circular template and
multiple random primers. The synthesis is initiated by multiple primers bound to the
template. DNA synthesis using strand displacement is carried out as in (B). However,
primers still present in the reaction mixture bind to the displaced strand and are used
as additional initiation points for DNA synthesis. The multiple products are long
concatemeric molecules of double-stranded DNA.
26
The application of RCA-technology for the discovery, characterization and
diagnosis of plant-infecting viruses has mainly concentrated on small single-stranded
DNA genomes of viruses in the families Geminiviridae (genome n=1 or 2, size 2.5-3
kbp) and Nanoviridae (genome n=6 to 8, size 1-1.5 kbp) (Johne et al., 2009). However,
James et al. (2011a) showed that it could be utilized for the detection and
characterization of larger genome sized viruses belonging to the family
Caulimoviridae, specifically the badnavirus, banana streak virus (BSV). Importantly,
James et al. (2011a) also demonstrated the utility of sequence-nonspecific phi29 based
RCA to differentiate between episomal sequences of BSV from the integrated forms
(EPRVs) present in some banana genotypes. Their study also showed that RCA could
be improved by the addition of target-specific primers. The optimized RCA protocol
was subsequently used for the characterization of badnaviruses infecting banana
(Baranwal et al., 2014; Carnelossi et al., 2014; James et al., 2011b; Javer-Higginson
et al., 2014; Wambulwa et al., 2012; Wambulwa et al., 2013), cacao (Chingandu et al.,
2017a, 2017b; Muller et al., 2018), fig (Laney et al., 2012), mulberry (Chiumenti et
al., 2016), Rubus spp. (Diaz-Lara et al., 2015) and yam (Bömer et al., 2018, 2016;
Sukal et al., 2017; Umber et al., 2014).
Over the last 10 years, next generation sequencing (NGS) has gained momentum
as a tool for viral whole genome characterization. It is considered to be a highly
efficient, rapid, low cost DNA, or RNA high-throughput sequencing option for plant
viruses genomes (Hadidi et al., 2016). Recently, RCA coupled with NGS has also
proven effective for the characterization of badnaviruses from cacao (Chingandu et al.,
2017a, 2017b; Muller et al., 2018).
27
2.8 Current knowledge of badnaviruses infecting yam
Yam-infecting badnaviruses are probably the most prevalent viruses infecting
yam globally (Bousalem et al., 2009; Eni et al., 2008a, 2008b; Kenyon et al., 2008).
Yam badnaviruses are transmitted mechanically and by several species of mealybugs
(family Pseudococcidae) in a semi-persistent manner (Atiri et al., 2003; Bömer et al.,
2016; Kenyon et al., 2001; Odu et al., 2004; Phillips et al., 1999). Badnavirus infection
in yam can be symptomless (Figure 6A) or cause a range of symptoms including veinal
chlorosis (Figure 6B), necrosis and distortions such as puckering and crinkling (Bömer
et al., 2016; Lebot, 2009; Phillips et al., 1999; Seal et al., 2014).
Badnaviruses were first reported infecting yams from the Caribbean, where
bacilliform-shaped virions were observed together with a flexuous virus, causing
internal brown spot disease in D. alata and D. cayenensis-rotundata (Harrison and
Roberts, 1973; Mantell and Haque, 1978). Two decades later the complete genome of
two DBV isolates were sequenced from Nigerian D. alata and named as Dioscorea
alata bacilliform virus (ICTV species name Dioscorea bacilliform AL virus, DBALV)
(Briddon et al., 1999; Phillips et al., 1999). Later, the complete genome sequences of
two additional isolates representing a second species were sequenced from D.
sansibarensis originating from Benin and named Dioscorea sansibarensis bacilliform
virus (ICTV species name Dioscorea bacilliform SN virus, DBSNV)(Seal and Muller,
2007).
28
Figure 6: Variable symptoms on yam leaves infected with badnaviruses. Leaves of
(A) D. rotundata (var. Ogoja) showing no marked viral symptoms and (B) D.
rotundata (var. Nwokpoko) showing veinal chlorosis (adapted from Seal et al., 2014).
29
Later, Kenyon et al. (2008) used degenerate badnavirus-specific primers which
amplify the core RT/RNase H-coding region to identify 11 new DBV sequence groups
(K1-K11) with <79% nucleotide identity to each other, together with two additional
sequence groups (K12-K13) with very low sequence similarity to other badnaviruses.
These latter two groups were considered by the authors to either represent highly
divergent badnaviruses, members of a new Caulimoviridae genus, or remnants of viral
sequences that had become integrated into the host genome following illegitimate
recombination. An additional DBV sequence group (DBV-D) from Guinea yam with
<80% similarity to previously characterized sequences was subsequently reported by
Bousalem et al. (2009). However, both of these studies used PCR-based approaches
with degenerate badnavirus primers and the existence of episomal virus counterparts
of these groups was not confirmed.
Bömer et al. (2016) used RCA and PCR to characterize three further sequence
groups (T13-T15) from yams and determined the complete genome sequence of two
of the groups, namely Dioscorea bacilliform rotundata virus 1 (ICTV species name
Dioscorea bacilliform RT virus 1 (DBRTV1), group T13) and Dioscorea bacilliform
rotundata virus 2 (ICTV species name Dioscorea bacilliform RT virus 2 (DBRTV2),
group T14). Bömer et al. (2018) and Umber et al. (2017) in separate studies published
complete genomes of two additional species, namely Dioscorea bacilliform rotundata
virus 3 (DBRTV3) and Dioscorea bacilliform trifida virus (ICTV species name
Dioscorea bacilliform TR virus (DBTRV)), respectively. DBV genome
characterization has received much attention in recent years due to the need for virus
testing of yam to facilitate germplasm exchange. However, most of these studies have
been restricted to the African and Caribbean region with the Pacific receiving very
little attention.
30
2.9 Conservation and utilization of yams
The International Institute of Tropical Agriculture (IITA) conserves the largest
collection of yams, 5788 yam accessions, which includes nine species with D.
rotundata and D. alata making up the majority of the collection (www.iita.org). Yam
germplasm collections are also maintained at the Central Tuber Crops Research
Institute (CTCRI) in Trivandrum (India), Vietnam Agricultural Science Institute
(VASI) in Hanoi (Vietnam), Phil Root Crops in Baybay (the Philippines), Vanuatu
Agricultural Research and Technical Centre (VARTC) Vanuatu, French National
Institute for Agricultural Research (INRA), Centre de Coopération Internationale en
Recherche Agronomique pour le Développement (CIRAD) in Guadeloupe (West
Indies) with additional smaller collections in China, Japan and many PICs. A
collection of PICs accessions is conserved as an in vitro collection at the SPC-CePaCT
in Fiji.
The collection at SPC-CePaCT was assessed by DNA fingerprinting using
simple sequence repeat (SSR) markers in 2011 by IITA and shown to contain a unique
subset of the global yam genetic diversity (SPC 2011, unpublished data). SPC-
CePaCT since has acquired other species of yam, including wild species, through
various donor-funded projects, in particular the Global Crop Diversity Trust to expand
its unique Pacific yam collection. SPC-CePaCT receives a long-term grant for
sustainable conservation of its yam collection and currently maintains an in vitro yam
collection (283 cultivars) which comprises seven species, namely, D. alata, D.
rotundata, D. esculenta, D. bulbifera, D. nummularia, D. transversa and D. trifida
(SPC 2018, unpublished data). The collection stands to expand as new species and
varieties are received from within and outside of the Pacific. However, as iterated
before, only 10% of the collection has been available for exchange due to the inability
31
to successfully certify the plants as virus-free due to the lack of diagnostic protocols.
2.10 Research problem and aim
Despite its significant value as a food security and climate resilient crop, and a
crop with potential for economic exploitation, yam production in the PIC’s remains
low. There is the potential to increase production through the introduction and
evaluation of new genetic diversity with important agronomical traits. This can be
achieved through utilization of field collections held in countries within the Pacific
region, the in vitro collection held at SPC-CePaCT and the collections held in other
global genebanks such as IITA. However, a lack of reliable diagnostic protocols for
the detection of yam badnaviruses has hindered the exchange of yam germplasm. The
African region has made great progress in characterizing the diversity of badnaviruses
in yam, however, very limited work has been done in the Pacific. Therefore, the aim
of this project was to identify and characterize yam-infecting badnaviruses in the
Pacific for the subsequent development of reliable diagnostic tests.
2.11 Objectives
The objectives of this study were therefore to:
1. Identify and characterize badnaviruses infecting Pacific collections of yam
germplasm.
2. Develop, evaluate and validate protocols for the detection of episomal
badnavirus infections in yam.
3. Investigate the genetic diversity of Pacific isolates of badnaviruses which
infect yam.
32
2.12 References
Abraham, K., Nair, G.P., 1991. Polyploidy and sterility in relation to sex in Dioscorea
alata L. (Dioscoreaceae). Genetica 83, 93–97.
Ali, M.M., Li, F., Zhang, Z., Zhang, K., Kang, D.-K., Ankrum, J.A., Le, X.C., Zhao,
W., 2014. Rolling circle amplification: a versatile tool for chemical biology,
materials science and medicine. Chem. Soc. Rev. 43, 3324.
Arnau, G., Abraham, K., Sheela, M.N.N., Chair, H., Sartie, A., Asiedu, R., Chair, H.,
Sartie, A., Asiedu, R., 2010. Yams, in: Bradshaw, J.E. (Ed.), Root and Tuber
Crops. Springer New York, New York, NY, pp. 127–148.
Arnau, G., Nemorin, A., Maledon, E., Abraham, K., 2009. Revision of ploidy status
of Dioscorea alata L. (Dioscoreaceae) by cytogenetic and microsatellite
segregation analysis. Theor. Appl. Genet. 118, 1239–1249.
Ashby, M.K., WaZrry, A., Bejarano, E.R., Khashoggi, A., Burrell, M., Lichtenstein,
C.P., 1997. Analysis of multiple copies of geminiviral DNA in the genome of
four closely related Nicotiana species suggest a unique integration event. Plant
Mol. Biol. 35, 313–321.
Asiedu, R., Sartie, A., 2010. Crops that feed the World 1. Yams. Food Secur. 2, 305–
315.
Atiri, G.I., Winter, S., Alabi, O.J., 2003. Yam, in: Loebenstein, G., Thottappilly, G.
(Eds.), Virus and Virus-like Diseases of Major Crops in Developing Countries.
Springer Netherlands, Dordrecht, The Netherlands, pp. 249–250.
Ayensu, E.S., Coursey, D.G., 1972. Guinea yams the botany, ethnobotany, use and
possible future of yams in West Africa. Econ. Bot. 26, 301–318.
Baranwal, V.K., Sharma, S.K., Khurana, D., Verma, R., 2014. Sequence analysis of
shorter than genome length episomal banana streak OL virus like sequences
33
isolated from banana in India. Virus Genes 48, 120–127.
Bejarano, E.R., Khashoggi, A, Witty, M., Lichtenstein, C., 1996. Integration of
multiple repeats of geminiviral DNA into the nuclear genome of tobacco during
evolution. Proc. Natl. Acad. Sci. U. S. A. 93, 759–764.
Bhat, A.I., Sasi, S., Revathy, K.A., Deeshma, K.P., Saji, K. V., 2014. Sequence
diversity among badnavirus isolates infecting black pepper and related species in
India. Virus Dis. 25, 402–407.
Bhattacharjee, R., Gedil, M., Sartie, A., Otoo, E., Dumet, D., Kikuno, H., Kumar, P.L.,
Asiedu, R., 2011. Dioscorea, Wild Crop Relatives: Genomic and Breeding
Resources, Industrial Crops. Springer Berlin Heidelberg, Berlin, Heidelberg.
Bömer, M., Rathnayake, A.I., Visendi, P., Silva, G., Seal, S.E., 2018. Complete
genome sequence of a new member of the genus Badnavirus, Dioscorea
bacilliform RT virus 3, reveals the first evidence of recombination in yam
badnaviruses. Arch. Virol. 163, 533–538.
Bömer, M., Turaki, A., Silva, G., Kumar, P., Seal, S., 2016. A sequence-independent
strategy for amplification and characterization of episomal badnavirus sequences
reveals three previously uncharacterized yam badnaviruses. Viruses 8, 188.
Bousalem, M., Arnau, G., Hochu, I., Arnolin, R., Viader, V., Santoni, S., David, J.,
2006. Microsatellite segregation analysis and cytogenetic evidence for tetrasomic
inheritance in the American yam Dioscorea trifida and a new basic chromosome
number in the Dioscoreae. Theor. Appl. Genet. 113, 439–451.
Bousalem, M., Durand, O., Scarcelli, N., Lebas, B.S.M., Kenyon, L., Marchand, J.L.,
Lefort, F., Seal, S.E., 2009. Dilemmas caused by endogenous pararetroviruses
regarding the taxonomy and diagnosis of yam (Dioscorea spp.) badnaviruses:
Analyses to support safe germplasm movement. Arch. Virol. 154, 297–314.
34
Briddon, R.W., Phillips, S., Brunt, A., Hull, R., 1999. Analysis of the sequence of
Dioscorea alata bacilliform virus; comparison to other members of the badnavirus
group. Virus Genes 18, 277–283.
Carnelossi, P.R., Bijora, T., Facco, C.U., Silva, J.M., Picoli, M.H.S., Souto, E.R.,
Oliveira, F.T. De, 2014. Episomal detection of banana streak OL virus in single
and mixed infection with Cucumber mosaic virus in banana “Nanicão Jangada.”
Trop. Plant Pathol. 39, 342–346.
Chabannes, M., Iskra-Caruana, M.-L., 2013. Endogenous pararetroviruses—a
reservoir of virus infection in plants. Curr. Opin. Virol. 3, 615–620.
Cheng, R.H., Olson, N.H., Baker, T.S., 1992. Cauliflower mosaic virus: A 420 subunit
(T=7), multilayer structure. Virology 186, 655–668.
Chingandu, N., Kouakou, K., Aka, R., Ameyaw, G., Gutierrez, O.A., Herrmann, H.-
W., Brown, J.K., 2017a. The proposed new species, cacao red vein virus, and
three previously recognized badnavirus species are associated with cacao swollen
shoot disease. Virol. J. 14, 199.
Chingandu, N., Zia-ur-rehman, M., Sreenivasan, T.N., Surujdeo-Maharaj, S.,
Umaharan, P., Gutierrez, O.A., Brown, J.K., Thyail, M.Z., Zia-ur-rehman, M.,
Sreenivasan, T.N., Surujdeo-Maharaj, S., Umaharan, P., Gutierrez, O.A., Brown,
J.K., Thyail, M.Z., 2017b. Molecular characterization of previously elusive
badnaviruses associated with symptomatic cacao in the New World. Arch. Virol.
162, 1363–1371.
Chiumenti, M., Morelli, M., De Stradis, A., Elbeaino, T., Stavolone, L., Minafra, A.,
2016. Unusual genomic features of a badnavirus infecting mulberry. J. Gen.
Virol. 97, 3073–3087.
Diaz-Lara, A., Mosier, N.J., Keller, K.E., Martin, R.R., 2015. A variant of Rubus
35
yellow net virus with altered genomic organization. Virus Genes 50, 104–110.
Elevitch, B.C.R., Love, K., 2011. Farm and Forestry Production and Marketing
Profiles: Highlighting value-added strategies., in: Elevitch, C.R. (Ed.), Specialty
Crops for Pacific Island Agroforestry. Permanent Agriculture Resources (PAR),
Holualoa, Hawai‘i., p. 14.
Eni, A.O., Hughes, J.D.A., Asiedu, R., Rey, M.E.C., 2008a. Sequence diversity among
badnavirus isolates infecting yam (Dioscorea spp.) in Ghana, Togo, Benin and
Nigeria. Arch. Virol. 153, 2263–2272.
Eni AO, Hughes JDA, Rey MEC, 2008b. Survey of the incidence and distribution of
five viruses infecting yams in the major yam-producing zones in Benin. Ann Appl
Biol 153, 223–32.
Essad, S., 1984. Geographic variation of basic chromosome numbers and polyploidy
in the Dioscorea genus with regard to counting for transversa Brown,
pilosiuscula Bert. and trifida (L.). Agronomie 4, 611–617.
FAOSTAT, 2018. Production Statistics (FAOSTAT). Rome, Italy: Food and
Agriculture Organization of the United Nations.
Filloux, D., Murrell, S., Koohapitagtam, M., Golden, M., Julian, C., Galzi, S., Uzest,
M., Rodier-Goud, M., D’Hont, A., Vernerey, M.S., Wilkin, P., Peterschmitt, M.,
Winter, S., Murrell, B., Martin, D.P., Roumagnac, P., 2015. The genomes of
many yam species contain transcriptionally active endogenous geminiviral
sequences that may be functionally expressed. Virus Evol. 1, vev002.
Fuji, S., Mitobe, I., Nakamae, H., Natsuaki, K.T., 1999. Nucleotide sequence of coat
protein gene of yam mild mosaic virus. J. Gen. Virol. 1415–1419.
Gamiette, F., Bakry, F., Ano, G., 1999. Ploidy determination of some yam species
(Dioscorea spp.) by flow cytometry and conventional chromosomes counting.
36
Genet. Resour. Crop Evol. 46, 19–27.
Geering, A.D., 2014. Caulimoviridae (Plant Pararetroviruses), in: ELS. John Wiley &
Sons, Ltd, Chichester, UK.
Geering, A.D.W., Hull, R., 2012. Family Caulimoviridae, in: King, A.M.Q., Adams,
M.J., Carstens, E.B., Lefkowitz, E.J. (Eds.), Virus Taxonomy. Ninth Report of
the International Committee on Taxonomy of Viruses. Elsevier Academic Press,
Amsterdam, The Netherlands, pp. 429–443.
Geering, A.D.W., Scharaschkin, T., Teycheney, P., 2010. The classification and
nomenclature of endogenous viruses of the family Caulimoviridae. Arch. Virol.
155, 123–131.
Girma, G., Spillane, C., Gedil, M., 2016. DNA barcoding of the main cultivated yams
and selected wild species in the genus Dioscorea. J. Syst. Evol. 54, 228–237.
Govaerts, R., Wilkin, P., Saunders, R., 2007. World checklist of Dioscoreales: yams
and their allies, Kew: Royal. ed. London, U.K.
Guimarães, K.M.C., Silva, S.J.C., Melo, A.M., Ramos-Sobrinho, R., Lima, J.S.,
Zerbini, F.M., Assunção, I.P., Lima, G.S.A., 2015. Genetic variability of
badnaviruses infecting yam (Dioscorea spp.) in northeastern Brazil. Trop. Plant
Pathol. 40, 111–118.
Hadidi, A., Flores, R., Candresse, T., Barba, M., 2016. Next-generation sequencing
and genome editing in plant virology. Front. Microbiol. 7, 1–12.
Harper, G., Hart, D., Moult, S., Hull, R., Geering, A., Thomas, J., 2005. The diversity
of banana streak virus isolates in Uganda. Arch. Virol. 150, 2407–2420.
Harper, G., Osuji, J.O., Heslop-Harrison, J.S., Hull, R., 1999. Integration of banana
streak badnavirus into the Musa genome: molecular and cytogenetic evidence.
Virology 255, 207–213.
37
Harrison, B.D.D., Roberts, I.M.M., 1973. Association of virus-like particles with
internal brown spot of yam (Dioscorea alata). Trop. Agric. 50, 355–340.
Huang, Q., Hartung, J.S., 2001. Cloning and sequence analysis of an infectious clone
of citrus yellow mosaic virus that can infect sweet orange via agrobacterium-
mediated inoculation. J. Gen. Virol. 82, 2549–2558.
Hull, R., 1996. Molecular biology of rice tungro viruses. Annu. Rev. Phytopathol. 34,
275–297.
James, A.P., Geijskes, R.J., Dale, J.L., A., Harding, R.M., 2011a. Development of a
novel rolling-circle amplification technique to detect banana streak virus that also
discriminates between integrated and episomal virus sequences. Plant Dis. 95,
57–62.
James, A.P., Geijskes, R.J., Dale, J.L., Harding, R.M., 2011b. Molecular
characterization of six badnavirus species associated with leaf streak disease of
banana in East Africa. Ann. Appl. Biol. 158, 346–353.
Javer-Higginson, E., Acina-Mambole, I., González, J.E., Font, C., González, G.,
Echemendía, A.L., Muller, E., Teycheney, P.Y., 2014. Occurrence, prevalence
and molecular diversity of banana streak viruses in Cuba. Eur. J. Plant Pathol.
138, 157–166.
Johne, R., Mu, H., Rector, A., Ranst, M. Van, Stevens, H., 2009. Rolling-circle
amplification of viral DNA genomes using phi29 polymerase. Trends Microbiol.
17, 205–211.
Kenyon, L., Lebas, B.S.M., Seal, S.E., 2008. Yams (Dioscorea spp.) from the South
Pacific Islands contain many novel badnaviruses: implications for international
movement of yam germplasm. Arch. Virol. 153, 877–889.
Kenyon, L., Shoyinka, S. a, Hughes, J. d’A., Odu, B.O., 2001. An overview of viruses
38
infecting Dioscorea yams in sub-Saharan Africa, in: Proceeding of Plant
Virology in Sub-Saharan Africa Conference. International institute of tropical
agricultural (IITA), Ibadan, pp. 432–439.
Kreuze, J.F., Perez, A., Untiveros, M., Quispe, D., Fuentes, S., Barker, I., Simon, R.,
2009. Complete viral genome sequence and discovery of novel viruses by deep
sequencing of small RNAs: A generic method for diagnosis, discovery and
sequencing of viruses. Virology 388, 1–7.
Laney, A.G., Hassan, M., Tzanetakis, I.E., 2012. An integrated badnavirus is prevalent
in fig germplasm. Phytopathology 102, 1182–1189.
Le Provost, G., Iskra-Caruana, M.-L., Acina, I., Teycheney, P.-Y., 2006. Improved
detection of episomal banana streak viruses by multiplex immunocapture PCR.
J. Virol. Methods 137, 7–13.
Lebot, V., 1997. Synthèse des résultats sur les plantes à tubercules en Nouvelle
Caledonie. Noumea, New Caledonia.
Lebot, V., 2009. Tropical Root and Tuber Crops: Cassava, Sweet Potato, Yams and
Aroids. Paris University, France.
Lebot, V., Malapa, R., Abraham, K., 2017. The Pacific yam (Dioscorea nummularia
Lam.), an under-exploited tuber crop from Melanesia. Genet. Resour. Crop Evol.
64, 217–235.
Lockhart, B.E.L., 1986. Purification and serology of a bacilliform virus associated
with banana streak disease. Phytopathology 76, 995-999.
Lockhart, B.E.L., Kiratiya-Angul, K., Jones, P., Eng, L., De Silva, P., Olszewski, N.E.,
Lockhart, N., Deema, N., Sangalang, J., 1997. Identification of piper yellow
mottle virus, a mealybug-transmitted badnavirus infecting Piper spp. in Southeast
Asia. Eur. J. Plant Pathol. 103, 303–311.
39
Malapa, R., Arnau, G., Noyer, J.L., Lebot, V., 2005. Genetic diversity of the greater
yam (Dioscorea alata L.) and relatedness to D. nummularia Lam. and D.
transversa Br. as revealed with AFLP markers. Genet. Resour. Crop Evol. 52,
919–929.
Mantell, S.H., Haque, S.Q., 1978. Incidence of internal brown spot disease in white
Lisbon yams (Dioscorea alata) during storage. Exp. Agric. 14, 167.
Menzel, W., Thottappilly, G., Winter, S., 2014. Characterization of an isometric virus
isolated from yam (Dioscorea rotundata) in Nigeria suggests that it belongs to a
new species in the genus Aureusvirus. Arch. Virol. 159, 603–606.
Mette, M.F., Kanno, T., Aufsatz, W., Jakowitsch, J., Winden, J. Van Der, Matzke,
M.A., Matzke, A.J.M., 2002. Endogenous viral sequences and their potential
contribution to heritable virus resistance in plants. The EMBO J. 21, 461–469.
Mignouna, H.D., Abang, M.M., Asiedu, R., 2008. Genomics of Yams, a Common
Source of Food and Medicine in the Tropics, in: Genomics of Tropical Crop
Plants. Springer New York, New York, NY, pp. 549–570.
Mignouna, H.D., Dansi, A., Zok, S., 2002. Morphological and isozymic diversity of
the cultivated yams (Dioscorea cayenensis/Dioscorea rotundata complex) of
Cameroon. Genet. Resour. Crop Evol. 49, 21–29.
Mignouna, H.D., Abang, Asiedu R, 2007. Advances in yam (Dioscorea spp.) genetics
and genomics, Proceedings of the 13th ISTRC Symposium. Arusha, Tanzania,
pp. 72–81.
Muller, E., Dupuy, V., Blondin, L., Bauffe, F., Daugrois, J.H., Nathalie, L., Iskra-
Caruana, M.L., 2011. High molecular variability of sugarcane bacilliform viruses
in Guadeloupe implying the existence of at least three new species. Virus Res.
160, 414–419.
40
Muller, E., Ravel, S., Agret, C., Abrokwah, F., Dzahini-Obiatey, H., Galyuon, I.,
Kouakou, K., Jeyaseelan, E.C., Allainguillaume, J., Wetten, A., 2018. Next
generation sequencing elucidates cacao badnavirus diversity and reveals the
existence of more than ten viral species. Virus Res. 244, 235–251.
Ndowora, T., Dahal, G., LaFleur, D., Harper, G., Hull, R., Olszewski, N.E., Lockhart,
B., 1999. Evidence that badnavirus infection in Musa spp. can originate from
integrated pararetroviral sequences. Virology 255, 214–220.
O’Sullivan, J.N., 2010. Yam nutrition and soil fertility management in the Pacific,
ACIAR Monograph. Canberra, Australia, pp. 122
Odu, B.O., Hughes, J.D.A.A., Asiedu, R., Ng, N.Q., Shoyinka, S.A., Oladiran, O.A.,
2004. Responses of white yam (Dioscorea rotundata) cultivars to inoculation
with three viruses. Plant Pathol. 53, 141–147.
Olszewski, N.E., Lockhart, B., 2011. Badnavirus, The Springer Index of Viruses.
Springer New York, New York, NY.
Phillips, S., Briddon, R.W., Brunt, A.A., Hull, R., 1999. The Partial Characterization
of a Badnavirus Infecting the Greater Asiatic or Water Yam (Dioscorea alata). J.
Phytopathol. 147, 265–269.
Ramser, J., Weising, K., Terauchi, R., Kahl, G., Lopez-Peralta, C., Terhalle, W., 1997.
Molecular marker based taxonomy and phylogeny of Guinea yam (Dioscorea
rotundata – D . cayenensis). Genome 40, 903–915.
Risimeri, J.B., 2001. Yams and food security in the lowlands of PNG, in: Food
Security for Papua New Guinea. Australian Centre for International Agricultural
Research, Canberra.
Scarcelli, N., Daïnou, O., Agbangla, C., Tostain, S., Pham, J.L., 2005. Segregation
patterns of isozyme loci and microsatellite markers show the diploidy of African
41
yam Dioscorea rotundata (2n=40). Theor. Appl. Genet. 111, 226–232.
Seal, S., Muller, E., 2007. Molecular analysis of a full-length sequence of a new yam
badnavirus from Dioscorea sansibarensis. Arch. Virol. 152, 819–825.
Seal, S., Turaki, A., Muller, E., Kumar, P.L., Kenyon, L., Filloux, D., Galzi, S., Lopez-
Montes, A., Iskra-Caruana, M.L., 2014. The prevalence of badnaviruses in West
African yams (Dioscorea cayenensis-rotundata) and evidence of endogenous
pararetrovirus sequences in their genomes. Virus Res. 186, 144–154.
SPYN, 2003. Yam : Cultivar Selection for Disease Resistance & Commercial Potential
in Pacific Islands, EU-INCO-DC project final report. Montpellier.
Staginnus, C., Iskra-Caruana, M.L., Lockhart, B., Hohn, T., Richert-Pöggeler, K.R.,
2009. Suggestions for a nomenclature of endogenous pararetroviral sequences in
plants. Arch. Virol. 154, 1189–1193.
Staginnus, C., Richert-Pöggeler, K.R., 2006. Endogenous pararetroviruses: two-faced
travelers in the plant genome. Trends Plant Sci. 11, 485–491.
Sukal, A., Kidanemariam, D., Dale, J., James, A., 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 Res. 238,
29–34.
Sukal, A.C., Taylor, M., Tuia, V.S., 2015. Viruses and their impact on the utilization
of plant genetic resources in the Pacific. Acta Hortic. 1101, 127–132.
Terauchi, R., Chikaleke, V.A., Thottappilly, G., Hahn, S.K., 1992. Origin and
phylogeny of Guinea yams as revealed by RFLP analysis of chloroplast DNA and
nuclear ribosomal DNA. Theor. Appl. Genet. 83, 743–751.
Umber, M., Filloux, D., Muller, E., Laboureau, N., Galzi, S., Roumagnac, P., Pavis,
C., Teycheney, P.Y., Seal, S.E., Iskra-Caruana, M.L., Pavis, C., Teycheney, P.Y.,
42
Seal, S.E., 2014. The genome of African yam (Dioscorea cayenensis-rotundata
complex) hosts endogenous sequences from four distinct badnavirus species.
Mol. Plant Pathol. 15, 790–801.
Umber, M., Gomez, R.M., Gélabale, S., Bonheur, L., Pavis, C., Teycheney, P.Y.,
2017. The genome sequence of Dioscorea bacilliform TR virus, a member of the
genus Badnavirus infecting Dioscorea spp., sheds light on the possible function
of endogenous Dioscorea bacilliform viruses. Arch. Virol. 162, 517–521.
Wambulwa, M.C., 2012. Rolling circle amplification is more sensitive than PCR and
serology-based methods in detection of banana streak virus in Musa germplasm.
Am. J. Plant Sci. 03, 1581–1587.
Wambulwa, M.C., Wachira, F.N., Karanja, L.S., Kiarie, S.M., Muturi, S.M., 2013.
The influence of host and pathogen genotypes on symptom severity in banana
streak disease. African J. Biotechnol. 12, 27–31.
Wilkin, P., Schols, P., Chase, M.W., Chayamarit, K., Furness, C. A., Huysmans, S.,
Rakotonasolo, F., Smets, E., Thapyai, C., 2005. A plastid gene phylogeny of the
yam genus, Dioscorea: roots, fruits and Madagascar. Syst. Bot. 30, 736–749.
Yang, I.C., Hafner, G.J., Dale, J.L., Harding, R.M., 2003a. Genomic characterization
of taro bacilliform virus. Arch. Virol. 148, 937–949.
Yang, I.C., Hafner, G.J., Revill, P.A., Dale, J.L., Harding, R.M., 2003b. Sequence
diversity of South Pacific isolates of Taro bacilliform virus and the development
of a PCR-based diagnostic test. Arch. Virol. 148, 1957–1968.
43
Chapter 3
Characterization of badnaviruses infecting Dioscorea spp.
in the Pacific reveals two putative novel species and the first
report of dioscorea bacilliform RT virus 2
Amit C. Sukal1, Dawit B. Kidanemariam1,2, James L. Dale1, Anthony P. James1,
Robert M. Harding1
1 Centre for Tropical Crops and Biocommodities, Queensland University of
Technology, Brisbane, 4001, Australia
2 National Agricultural Biotechnology Research Center, Ethiopian Institute of
Agricultural Research, P.O. Box 2003, Addis Ababa, Ethiopia
Virus Research 238:29–34
44
QUT Verified Signature
QUT Verified Signature
QUT Verified Signature
QUT Verified Signature
QUT Verified Signature
45
Abstract
The complete genome sequences of three new badnaviruses associated with yam
(Dioscorea spp.) originating from Fiji, Papua New Guinea and Samoa were
determined following rolling circle amplification of the virus genomes. The full-length
genomes consisted of a single molecule of circular double-stranded DNA of 8106 bp
for isolate FJ14, 7871 bp for isolate PNG10 and 7426 bp for isolate SAM01. FJ14 and
PNG10 contained three open reading frames while SAM01 had an additional open
reading frame which partially overlapped the 3′ end of ORF 3. Amino acid sequence
analysis of ORF 3 from the three isolates confirmed the presence of conserved motifs
typical of other badnaviruses. Phylogenetic analysis revealed the sequences to be
closely related to other Dioscorea–infecting badnaviruses. FJ14 and PNG10 appear to
be new species, which we have tentatively named dioscorea bacilliform ES virus
(DBESV) and dioscorea bacilliform AL virus 2 (DBALV2), respectively, while
SAM01 represents a Pacific isolate of the recently published dioscorea bacilliform RT
virus 2 and is described as dioscorea bacilliform RT virus 2-[4RT] (DBRTV2-[4RT]).
Keywords: Yam, Episomal badnavirus, rolling circle amplification, Dioscorea,
Dioscorea bacilliform ES virus, Dioscorea bacilliform AL virus 2
46
Members of the genus Badnavirus (family Caulimoviridae) have non-
enveloped, bacilliform-shaped virions with an approximate diameter of 30 nm and
length ranging from 120 to 150 nm (King et al., 2012). The genome consists of a single
molecule of circular, double-stranded DNA of 7.2–9.2 kb, typically encoding three
open reading frames (ORFs) all on the (+) strand (Geering, 2014). Replication occurs
via reverse transcription of a greater-than-genome length RNA which subsequently
serves as template for both the translation of viral proteins and reverse transcription
for replication of the genome (King et al., 2012). Badnavirus ORF 1 encodes a small
protein with an unknown function, while ORF 2 encodes a protein referred to as VAP
(virion-associated protein) which possesses a conserved coiled-coil motif (Stavolone
et al., 2001). ORF 3 encodes a large polyprotein that is cleaved into several mature
proteins, including a movement protein (MP), coat protein (CP), aspartic protease
(AP), reverse transcriptase (RT) and ribonuclease H (RNase H) (Geering, 2014).
Badnaviruses are transmitted through vegetative propagation, mealybug
vectors and in some cases through seed (Bhat et al., 2016). They are serologically and
genetically heterogeneous. Further, genomic DNA of several species, such as banana
streak viruses (BSV) (Gayral et al., 2008), dracaena mottle virus (DrMV) (Su et al.,
2007), fig badnavirus 1 (FBV-1) (Laney et al., 2012), and dioscorea bacilliform virus
(DBV) (Seal et al., 2014), are integrated into the host genome, which hinders the
development of diagnostic protocols (Kenyon et al., 2008; Seal et al., 2014). This
difficulty in diagnosis presents challenges to the safe exchange of germplasm.
Yams (Dioscorea spp.) are economically important, annual or perennial tuber-
bearing, dioecious, climbing, tropical monocots classified in the family Dioscoreaceae
(Mignouna et al., 2008). Cultivated yams are ranked as the fourth most important root
crop by production after potato, cassava and sweet potato (FAOSTAT, 2014). They
47
provide a staple food source for millions of people in Africa, South America, Asia and
the Pacific, and wild yams provide a valuable food source in times of famine. Yam
production is highest in West Africa, which accounts for 95% of the world’s total
production (Mignouna et al., 2008). Although most of the production occurs in the
African region, predominated by Dioscorea rotundata-cayenensis, yam is of
importance in the South Pacific where D. alata and D. esculenta are the dominant
species (Kenyon et al., 2008). Complete genomes of five Dioscorea-infecting
badnavirus species have been published, namely dioscorea bacilliform alata virus
(DBALV), dioscorea bacilliform sansibarensis virus (DBSNV), dioscorea bacilliform
rotundata virus 1 (DBRTV1), dioscorea bacilliform rotundata virus 2 (DBRTV2) and
dioscorea bacilliform trifida virus (DBTRV), however, none of these reports are from
the Pacific region (Briddon et al., 1999; Seal and Muller, 2007; Bömer et al., 2016,
Umber et al., 2016).
Yam production and improvement in the Pacific region is hindered by a lack
of genetic diversity. Germplasm exchange within the Pacific region and between
Pacific and Africa has been difficult due to a lack of reliable virus diagnostic protocols,
especially for badnaviruses. To address this problem, a project was initiated in 2014
by the Secretariat of the Pacific Community (SPC), Fiji, to characterize the diversity
of badnaviruses infecting yams in the Pacific region.
SPC maintains an in vitro collection of yams (278 cultivars) which is
comprised of seven different species, namely, D. alata, D. rotundata, D. esculenta, D.
bulbifera, D. nummularia, D. transversa and D. trifida. A subset of this collection (50
cultivars including D. alata [28], D. rotundata [1], D. esculenta [15], D. bulbifera [2],
D. nummularia [2], D. transversa [1] and D. trifida [1]) was initially screened using
an immunocapture polymerase chain reaction (IC-PCR) protocol with a general
48
badnavirus polyclonal antiserum (BenL) kindly provided by Prof. Ben Lockhart
(University of Minnesota, USA) and the degenerate badnavirus primers BadnaFP/RP
(Yang et al., 2003). In extracts from four of the 50 accessions, including two D. alata
types from Papua New Guinea (PNG) (DA-PNG03 and DA-PNG10), one D. esculenta
type from Fiji (DE-FJ14) and one D. rotundata type from Samoa (DR-SAM01), the
expected 579 bp product was amplified. To validate that the amplification was derived
from episomal badnavirus DNA and not integrated badnavirus sequences, total nucleic
acid (TNA) was extracted from leaf tissue (Kleinow et al., 2009) and subjected to
rolling circle amplification (RCA) using the TempliPhi 100 Amplification Kit (GE
Healthcare).
Briefly, 1 μl of TNA (adjusted to 500 ng/μl with sterile water) was mixed with
4 μl of the kit sample buffer and the mixture was denatured for 3 min at 95°C and snap
cooled on ice. Templihi kit reaction buffer (5 μl) was then mixed with 0.2 μl of phi29
DNA polymerase, added to each denatured TNA sample mixture, and incubated at
30°C for 18 h. The reaction mixture was then incubated at 65°C for 10 min to inactivate
the phi29 enzyme. Based on in-silico restriction analysis of published full-length
DBSNV (GenBank accession DQ822073 and DQ822074) sequences, the RCA
products were digested with the restriction enzymes BamHI and SalI for which the
published DBSNV sequences contained only one or two recognition sites.
Digestion of the RCA-amplified DNA using SalI resulted in a single fragment
of approximately 7.5 kb for all four samples, while digestion using BamHI yielded a
single fragment of approximately 7.5 kb from sample SAM01, two fragments of
approximately 4 and 3 kb from sample FJ14 and three fragments of approximately 3,
2.5 and 2 kb in samples PNG03 and PNG10. The restriction fragments were excised
and purified using Freeze ‘N Squeeze™ DNA Gel Extraction Spin Columns (Bio-Rad)
49
and subsequently ligated into appropriately cut and dephosphorylated pUC19 and
sequenced as described previously (James et al., 2011). BadnaFP/RP primers were
used to sequence the RT/RNase H–coding region.
Pairwise sequence comparison of the 529 bp RT/RNase H–coding region,
delimited by the BadnaFP/RP primers, of samples PNG03 and PNG10 revealed that
they were identical. When the PNG sequences were compared with the sequences from
FJ14 and SAM01 there was 64- 69% nucleotide similarity between the three groups.
When analysed using BLASTn, FJ14 was found to be most similar to DBALV
(accession KX008571) with 73.2% nucleotide similarity, while PNG03/PNG10 was
most similar to DBSNV (accession DQ822074) with 71% nucleotide similarity and
SAM01 was most similar to DBRTV2 (accession KX008577) with 95% nucleotide
similarity.
Since full genome sequences of the three putative badnaviruses were not
available in GenBank at the time of the original analysis, three independent full-length
clones of FJ14, PNG10 (as a representative of the PNG isolates) and SAM01,
generated from the SalI-digested RCA products, were completely sequenced in both
directions by primer walking. To confirm the sequences spanning the SalI restriction
sites, PCR was carried out using sequence-specific primers flanking this region.
Briefly, PCR master mix consisted of 10 μl of 2X GoTaq Green Master Mix
(Promega), 10 ρmol of each sequence-specific primer and 1 μl of TNA extract (diluted
to 30–50 ng/μl) in a final volume of 20 μl. PCR cycling conditions were as follows:
initial denaturation at 94°C for 2 min followed by 35 cycles of 94°C for 20 s, 50°C for
2 min, and 72°C for 2 min, with a final extension at 72°C for 10 min. The amplified
products were cloned into pGEM-T Easy (Promega) and sequenced as described
50
previously (James et al., 2011). Complete genome sequences were then assembled
using Geneious v9.0.2 (http://www.geneious.com; Kearse et al., 2012).
The assembled full-length genome sequences of FJ14, PNG10 and SAM01
comprised 8106 bp, 7871 bp and 7426 bp, respectively. The intergenic regions (IR) of
FJ14 and PNG10 comprised 1280 nt and 1210 nt, respectively, while the IR of SAM01
was considerably smaller at 751 nt. The IR of all isolates contained several conserved
nucleic acid motifs previously described for plant dsDNA viruses (Benfey and Chua,
1990; Medberry and Olszewski, 1993). A putative tRNAmet-binding site was identified
in all sequences (FJ141-18 and PNG101-18 - TGGTATCAGAGCTTGGTT, SAM011-18
- TGGTATCAGAGCTCGGTT; underlined nucleotides are mismatches) with 94%
and 89% nucleotide identity to the plant tRNAmet consensus sequence (3'
ACCAUAGUCUCGGUCCAA 5′), which has been previously described as the
priming site for reverse transcription (Medberry et al., 1990). The putative tRNAmet-
binding site was designated as the origin of the circular genome, consistent with the
convention currently used for badnaviruses. Transcriptional promoter elements
including putative TATA boxes and polyadenylation signals, analogous to the 35S
promotor of cauliflower mosaic virus, were also identified in the region upstream of
the tRNAmet-binding site (Table S1)
SnapGene® software (www.snapgene.com; GSL Biotech) was used to predict
the presence of putative ORFs on the plus strand of the three full-length sequences.
FJ14 and PNG10 were predicted to have three ORFs, while SAM01 was predicted to
have four ORFs, with the size and arrangement consistent with other published
badnavirus sequences (Fig. 1.). For FJ14, PNG10 and SAM01, ORF 1 was predicted
to encode a putative protein of 142 amino acids (aa) with a calculated Mr of 16.3 kDa,
16.5 kDa and 16.6 kDa, respectively. A conserved domain was identified within the
51
ORF 1 protein (pfam07028: DUF1319) (Finn et al., 2016), which is a member of
c106184 superfamily that appears to be restricted to badnaviruses (Marchler-Bauer et
al., 2015). ORF 2 of FJ14, PNG10 and SAM01 was predicted to encode a putative
protein of 128 aa (Mr=13.8 kDa), 131 aa (Mr=14.5 kDa) and 121 aa (Mr=13.6 kDa),
respectively. No conserved domains were identified in ORF 2 of any of the sequences.
ORF 3 of FJ14, PNG10 and SAM01 was predicted to encode a putative polyprotein of
2005 aa (Mr=226.6 kDa), 1946 aa (Mr=221.7 kDa) and 1892 aa (Mr=213.8 kDa),
respectively, with conserved domains for a movement protein, aspartic protease,
reverse transcriptase, ribonuclease H and RNA-binding zinc finger-like domain
(CXCX2CX4HX4C) predicted from the amino acid sequences.
In addition to the three typical ORFs found in badnaviruses, isolate SAM01
was predicted to have an additional ORF 4 of 417 nt (position 6656–7072), which
partially overlaps ORF 3 and encodes a 138 aa putative protein of calculated Mr of
15.5 kDa. The size and genome position of this ORF is similar to a putative small ORF
present in several other badnavirus genomes, including piper yellow mottle virus
(PYMoV) (Hany et al., 2014), pagoda yellow mosaic associated virus (PYMAV)
(Wang et al., 2014), yacon necrotic mottle virus (Lee et al., 2015), grapevine roditis
leaf discoloration-associated virus (GRLDaV) (Maliogka et al., 2015) and rubus
yellow net virus (Kalishuck et al., 2013). These small ORFs have little (5–20%) aa
sequence homology and no conserved domains.
52
Fig. 1. Linearized representation of the genome organization of (A) DBESV (isolate FJ14), (B) DBALV2 (isolate PNG10) and (C) DBRTV2-
[4RT] (isolate SAM01). The putative tRNAmet- binding site (tRNAmet), TATA box and polyadenylation signal (polyA); open reading frames (ORF)
1; ORF 2; ORF 3 showing movement protein (MP), capsid protein (CP), zinc finger (Zn), aspartic protease (AP), reverse transcriptase (RT) and
ribonuclease H (RNaseH) motifs.
53
To determine the taxonomic position of FJ14, PNG10 and SAM01,
phylogenetic analysis and sequence comparison to published yam badnavirus
sequences was carried out using the 529 bp RT/RNase H–coding region delimited by
the BadnaFP/RP primer binding sites. The sequences were aligned using the
CLUSTAL-W algorithm in MEGA7. A maximum likelihood method following
pairwise sequence comparison using the Kimura-2-parameter model was used to
construct a phylogenetic tree in MEGA7 (Kumar et al., 2016). This analysis showed
that FJ14, PNG10 and SAM01 cluster into three distinct putative species groups,
namely DeBV-A/K1, DeBV-B/K3 and T14, respectively (Fig. 2A) which is consistent
with previous phylogenetic analyses of characterized yam-infecting badnaviruses
(Kenyon et al., 2008; Bousalem et al., 2009; Eni et al., 2008; Seal et al., 2014; Bömer
et al., 2016; Umber et al., 2016). Phylogenetic analysis was also carried out using the
complete genome sequences of FJ14, PNG10, SAM01, badnaviruses infecting other
host plants as well as members of other genera within the family Caulimoviridae. This
analysis confirmed previous reports showing that yam-infecting badnaviruses cluster
with DBALV, DBTRV, DBRTV1, DBRTV2 and DBSNV in a single subgroup
(subgroup 4 in Fig. 2B) which is separate from badnaviruses infecting other hosts.
54
55
Fig. 2. A) Phylogenetic tree constructed using maximum likelihood method based on
the partial RT/RNaseH-coding sequence of DBESV (isolate FJ14), DBALV2 (isolate
PNG10) and DBRTV2-[4RT] (isolate SAM01) and previously described badnavirus
RT/RNaseH sequences from yam. Included in the analysis are (i) the monophyletic
groups described by Bousalem et al. (2009), where DBV-A=dioscorea bacilliform
virus A (A and B subgroups); DBV-B=dioscorea bacilliform virus B; DBV-
C=dioscorea bacilliform virus C; DBV-D=dioscorea bacilliform virus D; DeBV-
A=dioscorea esculenta bacilliform. virus A; DeBV-B=dioscorea esculenta bacilliform
virus B; DeBV-C=dioscorea esculenta bacilliform virus C; DeBV- D=dioscorea
esculenta bacilliform virus D; DeBV-E=dioscorea esculenta bacilliform E; DeBV-
56
F=dioscorea esculenta virus F; and DpBV=dioscorea pentaphylla bacilliform virus,
(ii) the 3 monophyletic groups (T13- T15) described by Bömer et al. (2016), (iii) the
one monophyletic group (U12) denoted by Umber et al. (2016), and (iv) the 11
monophyletic groupings (K1-K11) described by Kenyon et al. (2008). Equivalent
sequences from cacao swollen shoot virus (CSSV; NC_001574), banana streak OL
virus (BSOLV; AJ002234), commelina yellow mottle virus (ComYMV; X52938),
sugarcane bacilliform MO virus (SCBMOV; NC_008017), taro bacilliform virus
(TaBV; AF357836) and the outgroup rice tungro bacilliform virus (RTBV;
NC_001914; genus Tungrovirus) are also included. Boot- strap values for 1000
replicates with a cut-off value of 85%. Asterisks mark the groups for which full
genome representatives are available. B) Maximum likelihood phylogenetic tree
constructed from the alignment of complete genome sequences of DBESV (isolate
FJ14), DBALV2 (isolate PNG10), DBRTV2-[4RT] (isolate SAM01) and selected
badnaviruses. GenBank accession numbers are banana streak MY virus (BSMYV;
AY805074), banana streak VN virus (BSVNV; AY750155), banana streak GF virus
(BSGFV; AY493509), banana streak IM virus (BSIMV; HQ593112), banana streak
OL virus (BSOLV; AJ002234), banana streak UA virus (BSUAV; HQ593107),
banana streak UI virus (BSUIV; HQ593108), banana streak UL virus (BSULV;
HQ593109), banana streak UM virus (BSUMV; HQ593110), Bougainvillea chlorotic
vein banding virus (BCVBV; EU034539), commelina yellow mottle virus (ComYMV;
X52938), cacao swollen shoot virus (CSSV; NC_001574), citrus yellow mosaic virus
(CYMV; AF347695), dioscorea bacilliform AL virus (DBALV; X94578, X94580,
X94582, X94575), dioscorea bacilliform SN virus (DBSNV; DQ822073), dioscorea
bacilliform RT virus 1 (DBRTV1; KX008574), dioscorea bacilliform RT virus 2
(DBRTV2; KX008577), dioscorea bacilliform TR virus (DBTRV; KX430257), fig
57
badnavirus 1 (FBV-1; JF411989), grapevine roditis leaf discoloration-associated virus
(GRLDaV; HG940503), gooseberry vein banding associated virus (GVBaV;
JQ316114), grapevine vein clearing virus (GVCV; JF301669), pagoda yellow mosaic
associated virus (PYMAV; KJ013302), pineapple bacilliform CO virus (PBCOV;
GU121676), piper yellow mottle virus (PYMoV; KC808712), rubus yellow net virus
(RYNV; KM078034), sweet potato pakakuy virus (SPPV; FJ560943), sugarcane
bacilliform Guadeloupe D virus (SCBGDV; FJ439817), sugarcane bacilliform
Guadeloupe A virus (SCBGAV; FJ824813), sugarcane bacilliform IM virus
(SCBIMV; AJ277091), sugarcane bacilliform MO virus (SCBMOV; NC_008017),
taro bacilliform virus (TaBV; AF357836) and rice tungro bacilliform virus (RTBV;
NC_001914). RTBV (genus Tungrovirus) was used as the outgroup to the genus
Badnavirus. The topology of the tree supports the separation of badnaviruses into four
groups as depicted by Bömer et al. (2016) and Wang et al. (2014). Bootstrap values
for 1000 replicates with a cut-off value of 70% (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.).
58
The current species demarcation criteria for members of the genus Badnavirus
is a difference in the nucleotide sequence of the core polymerase (RT/RNase H-
coding) region of the genome of greater than 20% (King et al., 2012). Since the
nucleotide sequences of these regions in FJ14 and PNG10 differ from other published
full-length badnavirus sequences by more than 20%, these viruses probably represent
new species in the genus Badnavirus. To maintain the convention established by
previous authors who have described badnavirus species from Dioscorea spp. the
name dioscorea bacilliform esculenta virus (DBESV) and dioscorea bacilliform alata
virus 2 (DBALV2) is proposed for these putative new species from Fiji and PNG,
respectively. Although SAM01 was also considered a putative new species at the time
of analysis, this novel sequence from Samoa should now be considered as an isolate
(designated DBRTV2-[4RT]) of the recently characterized DBRTV2 (Bömer et al.,
2016) from D. rotundata, based on 95% nucleotide identity between the two sequences
in the RT/RNase H-coding region. The full-length sequence of DBESV (isolate FJ14),
DBALV2 (isolate PNG10) and DBRTV2-[4RT] (isolate SAM01) have been deposited
in the GenBank database under the accession numbers KY827394, KY827395 and
KY827393, respectively.
DBESV has high sequence similarity (>99.8%) with partial sequences
amplified using PCR from D. esculenta accessions from the Pacific (Fiji and PNG) as
well as 82–83% sequence similarity with partial sequences generated from D. alata
from PNG, Solomon Islands and Tonga (Kenyon et al., 2008; Fig. 2A sequence group
DeBV-A/K1). Similarly, DBALV2 has high sequence similarity (90–92%) with
partial sequences amplified using PCR from D. alata accessions from PNG and
Vanuatu and 82% sequence similarity with one sequence from D. alata from the
Philippines (Kenyon et al., 2008; Fig. 2A sequence group DeBV-B/K3). Interestingly,
59
additional sequences from these two subgroups were not detected in a subsequent
study investigating endogenous badnavirus sequences in yams (Bousalem et al., 2009).
Our results confirm the extant nature of two of these badnavirus groups through
sequencing of the episomal viral genome.
The present report confirms the occurrence of DBRTV2 in the Pacific, which
has previously only been recorded from the African region. Interestingly, previous
studies by Kenyon et al. (2008) and Bousalem et al. (2009) did not amplify sequences
with high similarity to DBRTV2. This may be due to the poor representation of D.
rotundata in the Pacific collection available to the former, or a result of the
geographical sources of accessions used in the latter study, with DBRTV2 only
previously reported in yam from Nigeria (Bömer et al., 2016).
As a preliminary study towards the development of diagnostic assays for these
three badnavirus species, PCR primers (Table S2) were designed based on the
sequences of DBESV and DBALV2 and using the consensus sequences of available
DBRTV2 isolates (isolates SAM01, KX008577, KX008578 and KX00857). These
primers were subsequently used in separate PCRs with TNA extracts from the 50 yam
accessions originally screened by IC-PCR using the BadnaFP/RP primers. PCR was
carried out using GoTaq Green, essentially as described previously, with primer
annealing temperatures listed in Table S2. Amplicons of the expected size were only
generated in the four yam samples (PNG03, PNG10, FJ14, SAM01) that had
previously tested positive for episomal badnavirus by IC-PCR and RCA, with
sequencing of the amplicons confirming their identity. Although these results suggest
that the primers may be suitable for use in diagnostic PCR assays for DBESV,
DBALV2 and DBRTV2-[4RT], further work is necessary to assess the genomic
sequence variability of these viruses in order to design the most appropriate primers
60
for virus detection. Badnaviruses are known to be highly variable at the genomic level
with Kenyon et al. (2008) reporting nucleotide differences of up to 18.0% and 16.3%
in the RT/RNase H-coding region of DBESV and DBALV2 isolates, respectively.
Importantly, this initial PCR screening work suggests that integrated sequences of
these three species are not present in the genomes of the yam species tested.
Furthermore, BLAST analysis of Dioscorea spp. expressed sequence tag (EST)
sequences available in GenBank, using the complete nucleotide sequences of DBESV,
DBALV2 and DBRTV2-[4RT], failed to identify any published sequences with
significant nucleotide sequence identity to these viruses, providing further evidence
that endogenous counterparts of DBESV, DBALV2 and DBRTV2 are not present in
D. alata, D. esculenta or in other Dioscorea spp. studied to date.
The recent increase in availability of full-length episomal sequences will
improve the clarity around the endogenous or exogenous nature of yam badnaviruses
and will help in the development of reliable diagnostics for badnaviruses in yam, thus
enabling safe international exchange of yam germplasm.
Conflict of interest
The authors declare no conflict of interest.
Financial support
The funding for the project was provided by the Australian Centre for
International Agricultural Research (#PC/2010/065). AS is a John Allwright
Fellowship recipient.
61
Acknowledgements
The authors would like to thank the Secretariat of the Pacific Community for
making their yam collections available for this project.
References
Bömer, M., Turaki, A.A., Silva, G., Kumar, P.L., Seal, S.E., 2016. A sequence-
independent strategy for amplification and characterization of episomal
badnavirus sequences reveals three previously uncharacterized yam
badnaviruses. Viruses 8, 188.
Benfey, P.N., Chua, N.H., 1990. The cauliflower mosaic virus 35S promoter:
combinatorial regulation of transcription in plants. Science 250, 959–966.
Bhat, A.I., Hohn, T., Selvarajan, R., 2016. Badnaviruses: the current global scenario.
Viruses 8, 177.
Bousalem, M., Durand, O., Scarcelli, N., Lebas, B.S.M., Kenyon, L., Marchand, J.-L.,
Lefort, F., Seal, S.E., 2009. Dilemmas caused by endogenous pararetroviruses
regarding the taxonomy and diagnosis of yam (Dioscorea spp.) badnaviruses:
analyses to support safe germplasm movement. Arch. Virol. 154, 297–314.
Briddon, R.W., Phillips, S., Brunt, A., Hull, R., 1999. Analysis of the sequence of
Dioscorea alata bacilliform virus; Comparison to other members of the
badnavirus group. Virus Genes 18, 277–283.
Eni, A.O., Hughes, J.D., Asiedu, R., Rey, M.E.C., 2008. Sequence diversity among
badnavirus isolates infecting yam (Dioscorea spp.) in Ghana, Togo, Benin and
Nigeria. Arch. Virol. 153, 2263–2272.
FAOSTAT, 2014, Rome, Italy http://faostat.fao.org.
62
Finn, R.D., Coggill, P., Eberhardt, R.Y., Eddy, S.R., Mistry, J., Mitchell, A.L., Potter,
S.C., Punta, M., Qureshi, M., Sangrador-Vegas, A., Salazar, A.G., Tate, J.,
Bateman, A., 2016. The Pfam protein families database: towards a more
sustainable future. Nucleic Acids Res. 44, D279–D285.
Gayral, P., Noa-Carrazana, J.-C., Lescot, M., Lheureux, F., Lockhart, B.E.L.,
Matsumoto, T., Piffanelli, P., Iskra-Caruana, M.-L., 2008. A single banana
streak virus integration event in the banana genome as the origin of infectious
endogenous pararetrovirus. J. Virol. 82, 6697–6710.
Geering, A.D., 2014. Caulimoviridae (Plant pararetroviruses). John Wiley&Sons, Ltd,
Chichester, UK.
Hany, U., Adams, I.P., Glover, R., Bhat, A.I., Boonham, N., 2014. The complete
genome sequence of Piper yellow mottle virus (PYMoV). Arch. Virol. 159, 385–
388.
James, A.P., Geijskes, R.J., Dale, J.L., Harding, R.M., 2011. Development of a novel
rolling circle amplification technique to detect banana streak virus that also
discriminates between integrated and episomal virus sequences. Plant Dis. 95,
57–62.
Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S., Buxton,
S., Cooper, A., Markowitz, S., Duran, C., Thierer, T., Ashton, B., Mentjies, P.,
Drummond, A., 2012. Geneious Basic: an integrated and extendable desktop
software platform for the organization and analysis of sequence data.
Bioinformatics 28, 1647–1649.
Kenyon, L., Lebas, B.S.M., Seal, S.E., 2008. Yams (Dioscorea spp.) from the South
Pacific Islands contain many novel badnaviruses: implications for international
movement of yam germplasm. Arch. Virol. 153, 877–889.
63
King, A.M.Q., Adams, M.J., Carstens, E.B., Lefkowitz, E.J., 2012. Virus taxonomy.
In: Ninth Report of the International Committee on Taxonomy of Viruses. San
Diego. Elsevier, 1338 pp.
Kleinow, T., Nischang, M., Beck, A., Kratzer, U., Tanwir, F., Preiss, W., Kepp, G.,
Jeske, H., 2009. Three C-terminal phosphorylation sites in the Abutilon mosaic
virus movement protein affect symptom development and viral DNA
accumulation. Virology 390, 89–101.
Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: Molecular evolutionary genetics
analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1854–1874.
Laney, A.G., Hassan, M., Tzanetakis, I.E., 2012. An integrated badnavirus is prevalent
in fig germplasm. Phytopathol 102, 1182–1189.
Lee, Y.-J., Kwak, H.-R., Lee, Y.-K., Kim, M.-K., Choi, H.-S., Seo, J.-K., 2015.
Complete genome sequence of yacon necrotic mottle virus, a novel putative
member of the genus Badnavirus. Arch. Virol. 160, 1139–1142.
Maliogka, V.I., Olmos, A., Pappi, P.G., Lotos, L., Efthimiou, K., Grammatikaki, G.,
Candresse, T., Katis, N.I., Avgelis, A.D., 2015. A novel grapevine badnavirus is
associated with the Roditis leaf discoloration disease. Virus Res. 203, 47–55.
Marchler-Bauer, A., Derbyshire, M.K., Gonzales, N.R., Lu, S., Chitsaz, F., Geer,
L.Y., Geer, R.C., He, J., Gwadz, M., Hurwitz, D.I., Lanczycki, C.J., Lu, F.,
Marchler, G.H., Song, J.S., Thanki, N., Wang, Z., Yamashita, R.A., Zhang, D.,
Zheng, C., Bryant, S.H., 2015. CDD: NCBI's conserved domain database.
Nucleic Acids Res. 43, D222–D226.
Medberry, S.L., Olszewski, N.E., 1993. Identification of cis elements involved in
Commelina yellow mottle virus promoter activity. Plant J. 3, 619–626.
64
Medberry, S.L., Lockhart, B.E.L., Olszewski, N.E., 1990. Properties of Commelina
yellow mottle virus's complete DNA sequence, genomic discontinuities and
transcript suggest that it is a pararetrovirus. Nucleic Acids Res. 18, 5505–5513.
Mignouna, H.D., Abang, M.M., Asiedu, R., 2008. Genomics of yams, a common
source of food and medicine in the tropics. In: Moore, P.H., Ming, R. (Eds.),
Plant Genetics and Genomics: Crops and Models. Springer, New York, pp. 549–
570.
Seal, S., Muller, E., 2007. Molecular analysis of a full-length sequence of a new yam
badnavirus from Dioscorea sansibarensis. Arch. Virol. 152, 819–825.
Seal, S., Turaki, A., Muller, E., Kumar, P.L., Kenyon, L., Filloux, D., Galzi, S., Lopez-
Montes, A., Iskra-Caruana, M.L., 2014. The prevalence of badnaviruses in West
African yams (Dioscorea cayenensis-rotundata) and evidence of endogenous
pararetrovirus sequences in their genomes. Virus Res. 186, 144–154.
Stavolone, L., Herzog, E., Leclerc, D., Hohn, T., 2001. Tetramerization is a conserved
feature of the virion-associated protein in plant pararetroviruses. J. Virol. 75,
7739–7743.
Su, L., Gao, S., Huang, Y., Ji, C., Wang, D., Ma, Y., Fang, R., Chen, X., 2007.
Complete genomic sequence of Dracaena mottle virus, a distinct badnavirus.
Virus Genes 35, 423–429.
Umber, M., Gomez, R.-M., Gélabale, S., Bonheur, L., Pavis, C., Teycheney, P.Y.,
2016. The genome sequence of Dioscorea bacilliform TR virus a member of the
genus Badnavirus infecting Dioscorea spp., sheds light on the possible function
of endogenous Dioscorea bacilliform viruses. Arch. Virol. 162, 517–521.
65
Wang, Y., Cheng, X., Wu, X., Wang, A., Wu, X., 2014. Characterization of complete
genome and small RNA profile of pagoda yellow mosaic associated virus a novel
badnavirus in China. Virus Res. 188, 103–108.
Yang, I.C., Hafner, G.J., Dale, J.L., Harding, R.M., 2003. Genomic characterization
of taro bacilliform virus. Arch. Virol. 148, 937–949.
66
Supplementary Information
Table S1. Arrangement of genome features of DBESV (isolate FJ14), DBALV2 (isolate PNG10) and DBRTV2-[4RT] (isolate SAM01).
Virus
Genome ORF 1 ORF 2 ORF 3 ORF 4 Transcriptional elements
Length (bp) Length Start-stop
(frame) Amino Protein
Lengt
h
Start-stop
(frame) Amino Protein
Lengt
h
Start-stop
(frame) Amino Protein
Lengt
h
Start-stop
(frame)
Amin
o
Protei
n TATA-box
Poly(A)-
tail
(% G + C) (bp) (codon use) acids (kDa) (bp) (codon use) acids (kDa) (bp) (codon use) acids (kDa) (bp) (codon use) acids (kDa) (position) <gap> (position)
DBESV
(isolate FJ14)
8106 429 824-1252
(+2)
142 16.3 387 1249-1635
(+1)
128 13.8 6020 1632-7651
(+3)
2005 226.6 7956-7962 <127
>
8090-8094
(46.2) (atg-tga) (atg-tga) (atg-tga) ttcTATATAAgac ATAAA
DBALV2
(isolate PNG10)
7871 429 769-1197
(+1)
142 16.5 396 1194-1589
(+3)
131 14.5 5841 1589-5841
(+2)
1946 221.7 7694-7700 <93> 7794-7799
(42.4) (atg-tga) (atg-taa) (atg-tga) ctcTATATAAgct AATAAA
DBRTV2-
[4RT] (isolate
SAM01)
7426 429 398-829 (+2) 142 16.6 366 823-1188 (+1) 121 13.6 5679 1183-6863
(+3)
1892 213.9 417 6656-7072
(+2)
138 15.50 7255-7261 <88> 7350-7355
(43.9) (atg-tga) (atg-tga) (atg-tag) (atg-taa) gccTATATAAgta AATAAA
67
Table S2. PCR primers used for the detection of DBESV (isolate FJ14), DBALV2 (isolate PNG10) and DBRTV2-[4RT] (isolate SAM01).
Target virus Primer name Sequence (5'-3') Tm (℃) Amplicon size (bp)
DBRTV2-[4RT] (isolate SAM01) YBV_GPT14-FP1 TCTCGCAGTTTACATCGATGA
53 438 YBV_GPT14-RP1 TTRGGYGGGATCTCCARTTC
DBALV2 (isolate PNG10) DBALV2_FP1 RCAGGAGCATGAGCAGCATT
57 316 DBALV2_RP1 CATCCTCTTTTCGCCATTTGG
DBESV (isolate FJ14) DBESV_FP1 GTGAGCCAATTCTGAGGGCT
60 1027 DBESV_RP1 GGTGGYAGYTGCARRTCAGG
68
This page is intentionally left blank
69
Chapter 4
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
Amit Sukal1,2, Dawit B Kidanemariam1, James Dale1, Robert M. Harding1 and
Anthony James1*
1 Centre for Tropical Crops and Biocommodities, Queensland University of
Technology, Brisbane, Queensland, Australia
2 Centre for Pacific Crops and Trees, Pacific Community, Suva, Fiji.
* Corresponding author:
E-mail address: [email protected] (APJ)
PLoS ONE 13:1-12
70
QUT Verified Signature
QUT Verified Signature
QUT Verified Signature
QUT Verified Signature
QUT Verified Signature
71
Abstract
We have characterized the complete genome of a novel circular double-stranded
DNA virus, tentatively named Dioscorea nummularia-associated virus (DNUaV),
infecting Dioscorea nummularia originating from Samoa. The genome of DNUaV
comprised 8139 bp and contained four putative open reading frames (ORFs). ORFs 1
and 2 had no identifiable conserved domains, while ORF 3 had conserved motifs
typical of viruses within the family Caulimoviridae including coat protein, movement
protein, aspartic protease, reverse transcriptase and ribonuclease H. A transactivator
domain, similar to that present in members of several caulimoviridae genera, was also
identified in the putative ORF 4. The genome size, organization, and presence of
conserved amino acid domains are similar to other viruses in the family
Caulimoviridae. However, based on nucleotide sequence similarity and phylogenetic
analysis, DNUaV appears to be a distinct novel member of the family and may
represent a new genus within the family Caulimoviridae.
Introduction
Yams (Dioscorea spp.) are ranked as the fourth most important root crop by
production after potato, cassava and sweet potato. They provide a staple food source
for millions of people in Africa, the Caribbean, South America, Asia and the Pacific
(1) while wild yams provide a valuable food source in times of famine. Yam
production is highest in West Africa, which accounts for 95% of the world’s total
production (2). Although most of the production occurs in the African region,
predominated by Dioscorea rotundata-cayenensis, yam is of importance in South
Pacific countries where D. alata and D. esculenta are the dominant species (3) with
some scattered cultivation of D. rotundata, D. bulbifera, D. nummularia, D. transversa
72
and D. trifida throughout the region. Yam cultivation and improvement in the Pacific
faces many agronomical challenges including yield losses due to pests and diseases
(4,5). To help address these issues, as well as improve food security and facilitate
commercial agricultural opportunities in the Pacific region, access to germplasm from
the Pacific and other regions (such as Africa) is needed for possible exploitation. An
important collection of Pacific yam germplasm is conserved in tissue culture at the
Centre for Pacific Crops and Trees (CePaCT) of the Pacific Community (SPC), Suva,
Fiji. This collection, together with yam germplasm from the International Institute of
Tropical Agriculture (IITA) in West Africa, could hold the key to addressing the
problems faced with yam cultivation in the Pacific. However, like many other
vegetatively propagated crops such as sugarcane, banana, cassava, aroids and sweet
potato, yams are prone to virus infection and accumulation. Therefore, the
identification of viruses infecting the crop and the development of reliable diagnostic
tests is critical to facilitate the safe exchange and utilization of yam germplasm.
Viruses belonging to the families Alphaflexiviridae (genus Potexvirus),
Betaflexiviridae (genus Carlavirus), Bromoviridae (genus Cucumovirus),
Caulimoviridae (genus Badnavirus), Potyviridae (genus Macluravirus and Potyvirus),
Secoviridae (genus Comovirus and Fabavirus) and Tombusviridae (genus
Aureusvirus) are known to infect yams (6,7). Of these, viruses belonging to the family
Caulimoviridae remain the least studied and the most difficult to diagnose due to their
significant genetic variability and, in some cases, the presence of integrated viral
sequences in the host genome (8–10).
The family Caulimoviridae consists of eight genera of reverse transcribing,
double-stranded DNA (dsDNA)-containing plant viruses, which are primarily
distinguished from each other based on particle morphology and genome organization
73
(11,12). Six of the genera, namely Caulimovirus, Cavemovirus, Petuvirus,
Rosadnavirus, Soymovirus and Solendovirus have isometric virions that are 52 nm in
diameter, while two genera, Badnavirus and Tungrovirus, have bacilliform virions
with dimensions of 30 x 130 to 150 nm (11,13). All family members have a genome
size between 7.2 to 9.2 kb with the coding capacity on the plus-strand. To date, only
species belonging to the genus Badnavirus have been identified from yams, namely
Dioscorea bacilliform alata virus (DBALV), DBALV2, Dioscorea bacilliform
esculenta virus (DBESV), Dioscorea bacilliform rotundata virus 1 (DBRTV1),
DBRTV2, DBRTV3, Dioscorea bacilliform sansibarensis virus (DBSNV) and
Dioscorea bacilliform trifida virus (DBTRV) (9,14–18). In addition to these full-length
viral sequences, a large number of partial reverse transcriptase (RT)-ribonuclease H
(RNase H) sequences which cluster within numerous different monophyletic groups
have also been PCR-amplified from yam germplasm (3,9,19–22). While the majority
of these groups cluster within the genus Badnavirus, several groups do not cluster with
any recognized genera within the family Caulimoviridae (3,21). Whether these
sequences are derived from episomal or integrated viral sequences or from another
source such as retrotransposons is unknown since they were generated by PCR.
In 2014, a project was initiated to characterize the diversity of badnaviruses
infecting yams in the Pacific region. In this paper, we report the identification of a
putative new member of the family Caulimoviridae from yam, tentatively named
Dioscorea nummularia-associated virus (DNUaV). The genome properties and
organization of DNUaV are described and its relationship to other members of the
family Caulimoviridae is discussed.
74
Materials and methods
Plant material and nucleic acid extraction
CePaCT maintains an in vitro collection of yams (278 accessions) which is
comprised of seven different species: D. alata (n=193), D. rotundata (n=32), D.
esculenta (n=41), D. bulbifera (n=8), D. nummularia (n=2), D. transversa (n=1) and
D. trifida (n=1) originating from Africa (obtained from IITA, Ibadan, Nigeria), Papua
New Guinea (PNG), Vanuatu, New Caledonia, Federated States of Micronesia (FSM),
Samoa and Tonga. Following screenhouse acclimatization for three months leaf
samples from 173 plants representative of the collection were used in this study. Total
nucleic acid (TNA) was extracted using a CTAB protocol (23) from approximately
100 mg of fresh leaf tissue. The purified TNA was treated with RNase A (1 μg/μl) and
the concentration adjusted to 500 ng/μl with sterile nuclease-free water.
RCA and sequencing
RCA was done essentially as described previously (24). Briefly, 1 μl of TNA
extract was used as template in RCA using the TempliPhi™ 100 Amplification Kit
(GE Healthcare, UK) with the addition of 1 μl of 10 mM 3’-exonuclease-protected
degenerate badnavirus 96 primers BadnaFP/RP (25) to bias amplification towards
badnavirus DNA.
RCA products were independently digested with EcoRI, KpnI, SphI and StuI
restriction endonucleases which were selected following in silico restriction analysis
of published yam badnavirus genome sequences, or based on experimental experience,
to generate useful restriction profiles. Digested RCA products were electrophoresed
through 1% agarose gels at 100 V for 1 h. Restriction fragments of approximately full-
length genome size (7-8 kb) were excised and ligated into appropriately digested and
75
de-phosphorylated pUC19. Plasmids were first screened via restriction analysis to
ensure desired inserts were present, then subjected to Sanger sequencing using either
universal M13 primers or BadnaFP/RP primers. The resulting sequences were used to
query the National Centre for Biotechnology Information (NCBI) database
(www.ncbi.nlm.nih.gov) with the BLASTn and BLASTx search functions. Where
BLAST analysis yielded a match to viral sequences, primer walking using sequence-
specific primers was used to generate full-length sequences in both directions.
To confirm the sequences spanning putative restriction sites, PCR was carried
out using sequence-specific primers flanking the region. PCR mixes consisted of 10
μl of 2x GoTaq Green Master Mix (Promega, USA), 5 ρmol of each sequence-specific
primer and 1 μl of DNA extract (diluted to ~50 ng/μl) in a final volume of 20 μl. PCR
cycling conditions were as follows: initial denaturation at 94°C for 2 min followed by
35 cycles of 94°C for 20 s, 50°C for 30 s and 72°C for 2 min, with a final extension at
72°C for 10 min. Amplicons were cloned into pGEM®-T Easy (Promega, USA) and
sequenced with primers M13F/R as described previously.
Putative full-length sequences were assembled using Geneious v11.0.5 (26).
SnapGene® software (www.snapgene.com; GSL Biotech) and ORFfinder
(https://www.ncbi.nlm.nih.gov/orffinder/) were used to predict putative ORFs on the
plus-strand of the assembled full-length sequences. InterPro software was used to scan
protein databases for conserved domains (27), while BLASTn and BLASTx were used
to search for sequence homologies in GenBank.
76
Sequence comparisons and phylogenetic analyses
Pairwise sequence comparison (PASC) was done using sequences
corresponding to amino acid residues L269-R672 of the cauliflower mosaic virus
(CaMV) polymerase (pol) gene. This region includes the conserved motifs of the RT-
and RNase H-coding regions (28) and is currently used for the demarcation of species
in the family Caulimoviridae (12). Nucleotide or translated amino acid sequences were
aligned using ClustalW alignment in MEGA7 (29). Phylogenetic analyses were done
using the nucleotide sequences of either the 529 bp RT/RNase H-coding region
delineated by the BadnaFP/RP primer binding sites or the pol gene sequences
described above. Sequences were aligned using ClustalW and phylogenetic trees were
constructed using the maximum-likelihood method (Kimura-2-parameter model) in
MEGA7 with 1000 bootstrap replication.
Viral DNA detection
Specific primers DNUaV-ORF4-FP1 (5'-CCGGGTTGCCAGTACAGAAT-
3') and DNUaV-ORF4-RP1 (5'-CGTGAAGCACCCAAACCTTG-3') were designed
following sequence analysis to amplify a 450 bp region of the putative ORF 4
sequence. PCR was carried out using GoTaq Green essentially as described previously
using 57°C as the annealing temperature. Amplicons were cloned and sequenced as
described earlier.
77
Results
Identification of DNUaV
Of the 173 samples analysed, none of which showed symptoms, 35 yielded
restriction profiles indicative of the presence of badnaviruses. Restriction analysis of
RCA products derived from two Samoan D. nummularia accessions (DN/WSM-01
and DN/WSM-02) using SphI 148 and StuI, resulted in putative full-length products
(~8 kb), while KpnI gave no digest products and digestion using EcoRI resulted in a
number of products smaller than 3.5 kb. These profiles were inconsistent with those
expected for known yam-infecting badnaviruses based on analysis of full-length
sequences available in GenBank. Therefore, the putative full-length SphI digested
fragments were cloned and sequenced. Sequences (~700 bp) originating from the
termini of the ~8 kb SphI fragments from both samples showed no nucleotide
similarity with published viral sequences. However, BLASTx analysis revealed that
the putative amino acid sequence from one end of the cloned fragments had low (32%)
similarity to the ORF 1 protein of the badnavirus, cacao yellow vein-banding virus
(CYVBV), and 31% similarity to the ORF 1 protein of the tungrovirus, rice tungro
bacilliform virus. Sequencing of the cloned fragments was subsequently carried out
using the degenerate badnavirus primers BadnaFP/RP. Sequences (~700 bp) were only
obtained using primer BadnaFP, with BLASTn analysis revealing 73-75% identity
with two partial RT/RNase H-coding sequences of a Dioscorea bacilliform virus
derived from D. nummularia (GenBank accessions AM072692 and AM421696).
Since the sequences of the two 8 kb-SphI clones from isolates DN/WSM-01 and
DN/WSM-02 showed 99% nucleotide similarity, the complete genomic sequence of
only one isolate, DN/WSM-01, was determined. This sequence was obtained from
78
three independent clones using primer walking, and the presence of the single SphI
restriction site was confirmed through additional PCR analysis and sequencing.
Genome organization, sequence and phylogenetic analysis
The complete genomic sequence of the virus isolate derived from yam
accession DN/WSM-01 was 8139 bp in length and was deposited in GenBank under
the accession number MG944237. Consistent with the RFLP patterns observed, the
genome contained 5 EcoRI sites, single SphI and StuI sites, and no KpnI site. The
genome of isolate DN/WSM-01 contained four putative ORFs which comprised 450
nt (ORF 1), 384 nt (ORF 2), 4737 nt (ORF 3) and 1371 nt (ORF 4) (Fig 1). ORFs 1
and 2, and 2 and 3 overlapped, whereas ORFs 3 and 4 were separated by one
nucleotide. Whereas ORFs 1 and 2 had overlapping stop/start codons (atga), the
putative start codon of ORF 3 was located 47 nucleotides 5' of the ORF 2 stop codon
(Fig 1). ORF 2 was in a -1 translational reading frame relative to ORF 1, while ORF 3
was in a +1 translational reading frame relative to ORF 2. The genome contained one
large intergenic region (IR), between ORF 4 and ORF 1 which comprised 1247 nt and
contained a putative tRNAmet binding site (5'-TGGTATCAGAGCAATGGT-3') with
88% nucleotide similarity to the plant tRNAmet consensus sequence (3'-
ACCAUAGUCUCGGUCCAA-5'), which has been described as the priming site for
reverse transcription (30). This was designated as the origin of the circular genome,
consistent with the convention used for other caulimoviridae members. A TATA-box
(TATATAA7944-7950) and polyadenylation signal (AAAAAATAA7981-7989), analogous
to the 35S promotor of CaMV, were also identified in the region 5' of the tRNAmet site.
Analysis of the translated ORF sequences failed to identify any conserved
domains in ORFs 1 and 2. In contrast, comparative sequence analysis of ORF 3
79
revealed several functional domains shared by all members of the family
Caulimoviridae including aspartic protease (Ala933-Ile1045, IPR021109), zinc finger
(Cys703-Cys708, IPR001878), RT (Lys1187-Ile1348, IPR000477) and RNase H domains
(Ser1469-Ala1574, IPR002156). In addition, a conserved movement protein (MP)
domain corresponding to M1-E327 of CaMV ORF 2 protein, and a coat protein (CP)
domain corresponding to L261–N429 of the CaMV ORF 4 protein, were also identified.
A transactivator (TAV) domain (Tyr80-202 Thr122, pfam01693), similar to that
present in ORF 6 of caulimoviruses and soymoviruses, was identified in the putative
ORF 4 sequence (Fig 1 and 2A-E).
When the full-length genome sequence of isolate DN/WSM-01 was used for
BLAST analysis with the search restricted to viruses (taxid:10239), the highest
nucleotide identity (70%) was to a 263 bp and 186 bp region of the RT domain of two
members of the genus Badnavirus, namely DBRTV2 (accession KX008579) and
cacao swollen shoot virus (CSSV; accession KX592572.1), respectively. BLAST
analysis of the putative protein sequences encoded by ORFs 1-4 of isolate DN/WSM-
01 revealed highest similarity with the ORF 1 protein of CYVBV (32%), ORF 2
protein of taro bacilliform virus (32%), ORF 3 polyprotein of fig badnavirus 1 (41%),
while ORF 4 had 26% similarity to amino acids 1443 to 1544 of Piper DNA virus 1.
80
Fig 1. Schematic representation of the genome organization of Dioscorea nummularia-associated virus (DNUaV). Large arrows represent the
putative ORFs. Conserved protein domains are shown: dark blue=movement protein (MP) domain corresponding to M1-E327 of CaMV ORF 2
protein; green=the putative coat protein (CP) domain corresponding to L261–N429 of the CaMV ORF 4 protein; black=zinc finger (Zn);
red=pepsin-like aspartic protease (AP); orange=reverse transcriptase (RT); purple=RNase H (RH); light blue=transactivator (TAV) domain. The
tRNAmet binding site (tRNAmet) and regulatory sequences including TATA box (TATA) and polyadenylation signal (PolyA) are also shown. The
relative position of restriction sites based on the complete genome sequence are shown above the genome.
81
82
Fig 2. Amino acid sequence alignments of the conserved motifs in the proteins of the
type member of each genus in the family Caulimoviridae. The type member for each
genus within the family Caulimoviridae used for comparison is as follows:
Caulimovirus - cauliflower mosaic virus (CaMV;V00141), Badnavirus - Commelina
yellow mottle virus (ComYMV; X52938), Cavemovirus – cassava vein mosaic virus
(CsVMV; U59751), Petuvirus - Petunia vein clearing virus (PVCV; U95208),
Tungrovirus - rice tungro bacilliform virus (RTBV; NC001914), Rosadnavirus - rose
yellow vein virus (RYVV; JX028536), Soymovirus - soybean chlorotic mottle virus
(X15828), Solendovirus – tobacco vein clearing virus (TVCV; AF190123). Identical
(asterisk/bold font), conserved (colon) and weakly conserved (dot) residues among the
members of the family are indicated.
83
PASC using either nucleotide or translated amino acid sequences of the pol
gene revealed an identity of 42 to 58% or 27 to 53%, respectively, between DNUaV
and the type species for each genus in the family Caulimoviridae (Table 1).
Phylogenetic analysis using partial RT/RNase H-coding sequences showed that
DNUaV forms a distinct subgroup outside of the genus Badnavirus, together with
several published sequences (GenBank accessions KY555561, AM072692 and
AM421696) previously reported from yams (Fig 3A). A similar tree topology, with
DNUaV clustering separately from recognized caulimoviridae genera, was obtained
when pol nucleotide sequences from published full-length sequences were analyzed
(Fig 3B).
PCR screening for DNUaV
Using primers designed to amplify a 450 bp region of DNUaV ORF 4, the 173
samples used in this study were tested for DNUaV by PCR. The expected size
amplicon was only generated from the Samoan D. nummularia samples, DN/WSM-
01 and DN/WSM-02. Sequence analysis of the cloned PCR amplicons from the two
samples revealed 99% similarity to each other and to the DNUaV ORF 4 sequence
generated using RCA.
84
Table 1. Mean pairwise nucleotide (above diagonal) and amino acid (below diagonal) similarity between the pol gene of DNUaV and the type
members of the eight current genera within the family Caulimoviridaea
DNUaV CaMV ComYMV CsVMV PVCV RTBV RYVV SbCMV TVCV
DNUaV
48 58 48 45 54 47 45 47
CaMV 41
46 46 51 47 48 48 47
ComYMV 53 36
43 42 49 45 42 43
CsVMV 36 36 32
45 48 54 45 64
PVCV 32 42 29 32
43 46 44 44
RTBV 45 36 40 33 30
46 43 48
RYVV 39 40 35 39 34 35
44 51
SbCMV 32 39 28 27 29 27 32
43
TVCV 38 37 32 48 30 36 37 28
aAbbreviations for the type members of each genus are: Caulimovirus - cauliflower mosaic virus (CaMV; V00141), Badnavirus - Commelina
yellow mottle virus (ComYMV; X52938), Cavemovirus - cassava vein mosaic virus (CsVMV; U59751), Petuvirus - Petunia vein clearing virus
(PVCV;U95208), Tungrovirus - rice tungro bacilliform virus (RTBV; NC001914), Rosadnavirus – rose yellow vein virus (RYVV; JX028536),
Soymovirus - soybean chlorotic mottle virus (X15828), Solendovirus - tobacco vein clearing virus (TVCV; AF190123) used in the analysis above.
85
86
87
Fig 3. Phylogenetic analysis using the maximum-likelihood method following
ClustalW alignment in MEGA7 (29) to infer evolutionary relationships of DNUaV.
Bootstrap values (1,000 replicates) are shown above nodes when greater than 70%.
(A) Phylogenetic tree constructed using sequences of the RT/RNase H-coding region
delineated by the BadnaFP/RP primers (25). This analysis includes badnavirus
RT/RNase H-coding sequences identified from yams (3,9,19-22), badnaviruses
infecting other crops and the homologous region of other caulimoviridae members
(See S1 Table and S2 Table for complete list of sequences used in the analysis).
Subgroups representing published yam badnavirus sequences have been collapsed to
improve the presentation of the tree, while the badnavirus groups that have
representative full genome sequence available are marked with an asterisk (*); (B)
Phylogenetic tree using pol gene nucleotide sequences of DNUaV and representative
members of family Caulimoviridae (See S2 Table for list of sequences included in the
analysis). The pol gene sequences are equivalent to amino acid residues L269-R672 from
the translated ORF 5 nucleotide sequence of cauliflower mosaic virus (CaMV).
88
Discussion
In this study, we identified and characterized a novel DNA virus infecting D.
nummularia which we have tentatively named Dioscorea nummularia-associated virus
(DNUaV). Although the genome size and organization, and the presence of conserved
amino acid domains of DNUaV, is typical of other viruses in the family
Caulimoviridae, there are several molecular features of the virus that distinguish it
from the current genera.
The ICTV uses several criteria to classify members of the family
Caulimoviridae. The most common criterion for demarcation of species uses
differences in the nucleotide sequence of the pol gene (AP/RT/RNase H-coding
region) of more than 20%. Comparisons of the pol gene sequence of DNUaV with
other Caulimoviridae showed the highest identity (76%) to a partial sequence of
Dioscorea bacilliform virus isolate SB10a_Dn derived from D. nummularia (3). Based
on differences in the nucleotide sequence identity of more than 20%, DNUaV appears
to be a novel virus in the family Caulimoviridae.
In addition to nucleotide sequence similarity, distinctions between genera
within the family Caulimoviridae are also based on the type of host plant, particle
morphology, genome organization and the presence and arrangement of conserved
protein-coding motifs. DNUaV encodes four ORFs with the size of ORFs 1-3
consistent with both badnavirus and tungrovirus members, as are the arrangement of
the characteristic MP, CP, Zn-finger binding domain and the AP-RT-RNase H-coding
regions of ORF 3. The relative positions of ORF 1 and 2 are similar to those of
badnaviruses, while ORFs 2 and 3 overlap each other by 47 nt which is also similar to
the badnaviruses CSSV, gooseberry vein banding virus, Piper yellow mottle virus and
sweet potato pakakuy virus (31–33). However, unlike those badnaviruses with a fourth
89
ORF which always overlaps with ORF 3, ORF 4 of DNUaV is separated from ORF 3
by a short intergenic region which is more similar to genome organization of RTBV,
the sole member of the genus Tungrovirus. Further, the size of DNUaV ORF 4 is also
similar to that of RTBV. Unlike RTBV, however, the DNUaV ORF 4 gene product
contains a conserved translation transactivator domain, which is typical of ORF 6 of
caulimoviruses and soymoviruses, and which is also present in ORF 4 of
cavemoviruses and solendoviruses. However, unlike the DNUaV ORF 4 sequence, the
ORF 4 sequences of both cavemoviruses and solendoviruses also includes the
conserved coiled-coil motifs characteristic of the virion-associated protein. Clearly,
determination of virion morphology and whether infected plants contain inclusion
bodies typical of members of the genus Caulimovirus is required before the taxonomic
status of DNUaV can be fully resolved. However, based on the sequence information
presented, DNUaV appears to be a distinct, novel member of Caulimoviridae.
PASC carried out using pol gene sequences showed 42 to 58% nucleotide or
27 to 53% amino acid sequence identity between DNUaV and the type members of
each genus within the family Caulimoviridae (Table 1). This level of nucleotide
sequence identity is typical of that between the established genera within the family
Caulimoviridae, which ranges from 42 to 64% (Table 1). Further, the level of amino
acid sequence identity is similar to the range of 27 to 48% identity between the type
members of each genus. Of the eight type members included in the analysis DNUaV
shares the highest level of amino acid identity (53%) with ComYMV, the type member
of the genus Badnavirus (Table 1), suggesting that DNUaV is most closely related to
the badnaviruses. However, phylogenetic analyses using either partial RT/RNase H-
coding sequences (Fig 3A) or pol gene sequences (Fig 3B), indicates that DNUaV is
basal to, and distinct from, the badnaviruses, forming a distinct clade between the
90
single member of the genus Tungrovirus, RTBV, and the genus Badnavirus. This
suggests that DNUaV may belong in a new, distinct genus within the family
Caulimoviridae.
Previous studies investigating the occurrence of badnaviruses in yams have
reported large numbers of badnavirus partial RT/RNase H-coding (529 bp) sequences
generated using the BadnaFP/RP primers (3,9,10,18–22). Phylogenetic analyses of
these sequences identified four distinct sequence groups, namely, K12 and K13 (3)
and T16 and T17 (21), which clustered into two monophyletic groups (K12/T16 and
K13/T17) outside of the eight currently recognized genera within the family
Caulimoviridae. Our phylogenetic analysis revealed that DNUaV clusters with the
monophyletic group K12/T16 (Fig 3A). Since the sequences reported in these previous
studies were obtained using a PCR based approach, the authors were unable to confirm
their episomal nature and so theorized that the sequence groups could represent either
divergent badnaviruses, ancient endogenous pararetrovirus sequences, or possibly new
genera within the family Caulimoviridae. The full-length DNUaV sequence presented
here provides strong evidence that the sequences in group K12/T16 may also be
derived from episomal virus(es) infecting yam.
When the yam germplasm collection held at CePaCT was tested for DNUaV
using primers designed from DNUaV ORF 4, only 2/173 samples tested positive, both
of which were D. nummularia from Samoa. Sequencing of the PCR products from the
two accessions revealed 99% nucleotide sequence identity to the full-length RCA-
derived sequence, indicating that the sequence was conserved in both isolates. These
results suggest that DNUaV does not appear to be integrated into the genome of
Dioscorea spp. as the only two samples that tested positive with PCR also tested
positive using RCA. Sequences with high similarity to DNUaV have previously been
91
identified from D. nummularia originating from the Solomon Islands (3), however, we
were unable to obtain yam samples from the Solomon Islands for testing. The
distribution of DNUaV in the Pacific needs to be determined as the current sample set
included only two D. nummularia accessions, both from Samoa.
This research builds on the work carried out previously (3,17) in characterizing
caulimoviridae from yams in the Pacific and is important in confirming the episomal
nature of reported sequences. An understanding of the episomal virus diversity
infecting yam will enable genebanks to test their genetic resources to ensure safe
distribution. The diagnostic protocol described here for detecting DNUaV may be
suitable for routine diagnostic screening for DNUaV in yam germplasm collections.
Acknowledgments
The authors would like to thank the Centre for Pacific Crops and Trees (CePaCT) of
the Pacific Community (SPC) for making their yam collections available for this
project. Authors would also like to thank Dr. Michael Furlong and Dr. Grahame
Jackson for their support and advice on this research.
92
References
1. FAOSTAT, 2018. Production Statistics (FAOSTAT). Food and Agriculture
Organization of the United Nations. Rome.
2. Mignouna HD, Abang MM, Asiedu R. Genomics of yams, a common source of
food and medicine in the tropics. In: Moore PH, Ming R, editors. Genomics of
tropical crop plants. Springer, New York; 2008; 549–570.
3. Kenyon L, Lebas BSM, Seal SE. Yams (Dioscorea spp.) from the South Pacific
Islands contain many novel badnaviruses: implications for international
movement of yam germplasm. Arch Virol. 2008;153: 877–889.
4. SPYN. Yam: cultivar selection for disease resistance and commercial potential
in Pacific Islands. CIRAD, Montpellier; 2003.
5. Sukal AC, Taylor M, Tuia VS. Viruses and their impact on the utilization of
plant genetic resources in the Pacific. Acta Hortic. 2015;1101: 127–132.
6. Kenyon L, Shoyinka SA, Hughes Jd’A, Odu BO. An overview of viruses
infecting Dioscorea yams in sub-Saharan Africa. In: Hughes Jd’A, Odu BO,
editors. Plant Virology in Sub-Saharan Africa: Proceedings of a conference
organized by IITA. International Institute of Tropical Agriculture, Ibadan; 2001:
432–439.
7. Menzel W, Thottappilly G, Winter S. Characterization of an isometric virus
isolated from yam (Dioscorea rotundata) in Nigeria suggests that it belongs to a
new species in the genus Aureusvirus. Arch Virol. 2014;159: 603–606.
8. Bhat A, Hohn T, Selvarajan R. Badnaviruses: The current global scenario.
Viruses. 2016;8: 177.
9. Bömer M, Turaki AA, Silva G, Kumar PL, Seal SE. A sequence-independent
strategy for amplification and characterization of episomal badnavirus
93
sequences reveals three previously uncharacterized yam badnaviruses. Viruses.
2016;8: 188.
10. Seal S, Turaki A, Muller E, Kumar PL, Kenyon L, Filloux D, et al. The
prevalence of badnaviruses in West African yams (Dioscorea cayenensis-
rotundata) and evidence of endogenous pararetrovirus sequences in their
genomes. Virus Res. 2014;186: 144–154.
11. Geering ADW. Caulimoviridae (Plant Pararetroviruses). In: Encyclopedia of
Life Sciences. John Wiley & Sons Ltd, Chichester; 2014.
12. Geering ADW, Hull R. Family Caulimoviridae. In: King AMQ, Adams MJ,
Carstens EB, Lefkowitz EJ, editors. Virus Taxonomy: Ninth report of the
international committee on taxonomy of viruses. Elsevier Academic Press,
Amsterdam; 2012: 429–443.
13. Hull R. Molecular biology of rice tungro viruses. Annu Rev Phytopathol.
1996;34: 275–297.
14. Bömer M, Rathnayake AI, Visendi P, Silva G, Seal SE. Complete genome
sequence of a new member of the genus Badnavirus, Dioscorea bacilliform RT
virus 3, reveals the first evidence of recombination in yam badnaviruses. Arch
Virol. 2017;163: 533-538.
15. Briddon RW, Phillips S, Brunt A, Hull R. Analysis of the sequence of Dioscorea
alata bacilliform virus; comparison to other members of the badnavirus group.
Virus Genes. 1999;18: 277–283.
16. Seal S, Muller E. Molecular analysis of a full-length sequence of a new yam
badnavirus from Dioscorea sansibarensis. Arch Virol. 2007;152: 819–825.
17. Sukal A, Kidanemariam D, Dale J, James A, Harding R. Characterization of
badnaviruses infecting Dioscorea spp. in the Pacific reveals two putative novel
94
species and the first report of Dioscorea bacilliform RT virus 2. Virus Res.
2017;238: 29–34.
18. Umber M, Gomez RM, Gélabale S, Bonheur L, Pavis C, Teycheney PY. The
genome sequence of Dioscorea bacilliform TR virus, a member of the genus
Badnavirus infecting Dioscorea spp., sheds light on the possible function of
endogenous Dioscorea bacilliform viruses. Arch Virol. 2017;162: 517–521.
19. Bousalem M, Durand O, Scarcelli N, Lebas BSM, Kenyon L, Marchand JL, et
al. Dilemmas caused by endogenous pararetroviruses regarding the taxonomy
and diagnosis of yam (Dioscorea spp.) badnaviruses: Analyses to support safe
germplasm movement. Arch Virol. 2009;154: 297–314.
20. Eni AO, Hughes JDA, Asiedu R, Rey MEC. Sequence diversity among
badnavirus isolates infecting yam (Dioscorea spp.) in Ghana, Togo, Benin and
Nigeria. Arch Virol. 2008;153: 2263–2272.
21. Turaki AA, Bömer M, Silva G, Kumar PL, Seal SE. PCR-DGGE analysis:
Unravelling complex mixtures of badnavirus sequences present in yam
germplasm. Viruses. 2017;9: 181.
22. Umber M, Filloux D, Muller E, Laboureau N, Galzi S, Roumagnac P, et al. The
genome of African yam (Dioscorea cayenensis-rotundata complex) hosts
endogenous sequences from four distinct badnavirus species. Mol Plant Pathol.
2014;15: 790–801.
23. Kleinow T, Nischang M, Beck A, Kratzer U, Tanwir F, Preiss W, et al. Three C-
terminal phosphorylation sites in the abutilon mosaic virus movement protein
affect symptom development and viral DNA accumulation. Virol. 2009;390: 89–
101.
95
24. James AP, Geijskes RJ, Dale JL, Harding RM. Development of a novel rolling-
circle amplification technique to detect banana streak virus that also
discriminates between integrated and episomal virus sequences. Plant Dis.
2011;95: 57–62.
25. Yang IC, Hafner GJ, Dale JL, Harding RM. Genomic characterization of taro
bacilliform virus. Arch Virol. 2003;148:937–949.
26. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, et al.
Geneious Basic: An integrated and extendable desktop software platform for the
organization and analysis of sequence data. Bioinformatics. 2012;28: 1647–
1649.
27. Finn RD, Attwood TK, Babbitt PC, Bateman A, Bork P, Bridge AJ, et al.
InterPro in 2017-beyond protein family and domain annotations. Nucleic Acids
Res. 2017;45: D190–D199.
28. Geering ADW, Scharaschkin T, Teycheney P-Y. The classification and
nomenclature of endogenous viruses of the family Caulimoviridae. Arch Virol.
2010;155: 123–131.
29. Kumar S, Stecher G, Tamura K. MEGA7: Molecular evolutionary genetics
analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016;33: 1870–1874.
30. Medberry SL, Lockhart BEL, Olszewski NE. Properties of commelina yellow
mottle virus’s complete DNA sequence, genomic discontinuities and transcript
suggest that it is a pararetrovirus. Nucleic Acids Res. 1990;18: 5505–5513.
31. Hany U, Adams IP, Glover R, Bhat AI, Boonham N. The complete genome
sequence of Piper yellow mottle virus (PYMoV). Arch Virol. 2014;159: 385–
388.
96
32. Kreuze JF, Perez A, Untiveros M, Quispe D, Fuentes S, B 425 arker I, et al.
Complete viral genome sequence and discovery of novel viruses by deep
sequencing of small RNAs: A generic method for diagnosis, discovery and
sequencing of viruses. Virol. 2009;388: 1–7.
33. Petrzik K, Přibylová J, Špak J. Molecular analysis of gooseberry vein banding
associated virus. Acta Virol. 2012;56: 119–124.
97
Supporting Information
S1 Table. Details of yam partial RT/RNase H-coding sequences used in the
phylogenetic analysis of DNUaV.
Phylogenetic
group Isolate GenBank accession Reference
DeBV-A/K1 SB42 Da AM072696 Kenyon et al., 2008
DBESV KY827394 Sukal et al., 2017
FJ75c De AM072663 Kenyon et al., 2008
DeBV-B/K2 PG137 De AM072682 Kenyon et al., 2008
PH06a De AM072688 Kenyon et al., 2008
DeBV-B/K3 PG141 Da AM072683 Kenyon et al., 2008
VU227 Da AM072704 Kenyon et al., 2008
DBALV2 KY827395 Kenyon et al., 2008
DsBV/K4 B394Ds DQ822073 Seal & Muller, 2007
B396Ds DQ822074 Seal & Muller, 2007
DBV-C/K5 FJ60b Dr AM072659 Kenyon et al., 2008
Mt9122Dt AM503398 Bousalem et al., 2009
NGl3841Dc KX008585 Bömer et al., 2016
NGb0005Da2 KX008581 Bömer et al., 2016
NGl1950Dr KX008589 Bömer et al., 2016
S1g6Dr KF829974 Umber et al., 2014
DeBV-D/K6 FJ65c De AM072661 Kenyon et al., 2008
PG180 De AM072687 Kenyon et al., 2008
DeBV-E/K7 PG110bDe AM072677 Kenyon et al., 2008 PH06b De AM072689 Kenyon et al., 2008
DBV-A(A)/K8 Gn502Dr AM503395 Bousalem et al., 2009
Gn845Dr AM503397 Bousalem et al., 2009
NGb0477Dr KX008586 Bömer et al., 2016
BfA103Dc AM503393 Bousalem et al., 2009
NGb0310Da2 KX008583 Bömer et al., 2016
DaBVa X94576, X94581 Briddon et al, 1999
NG3Da AM944573 Bömer et al., 2016
VU249 Db AM072705 Kenyon et al., 2008
DBALV-2ALb KX008594 Bömer et al., 2016
DBALV-2ALa KX008571 Bömer et al., 2016
NGb1844Dr2 KX008592 Bömer et al., 2016
NGb1892Dr1 KX008587 Bömer et al., 2016
DaBVb X94575, X94582 Briddon et al, 1999
NG1Da AM944571 Bömer et al., 2016
NGb0005Da1 KX008580 Bömer et al., 2016
NGb0310Da1 KX008582 Bömer et al., 2016
98
GH03 Dr AM072664 Bousalem et al., 2009
DBALV-3RT KX008595 Bömer et al., 2016
NG01 Dr AM072673 Bousalem et al., 2009
NGb1892Dr2 KX008588 Bömer et al., 2016
NGb2475Dr KX008590 Bömer et al., 2016
BN2Da AM944584 Eni et al., 2008
GHL2d Dr AM072668 Bousalem et al., 2009
DBV-B/K9 BN4Dr AM944586 Eni et al., 2008
Cu1Da AM503359 Bousalem et al., 2009
DBTRV KX430257 Umber et al., 2017
Bf1052Dr AM503363 Bousalem et al., 2009
BfA102Dr AM503365 Bousalem et al., 2009
FJ60a Dr AM072658 Kenyon et al., 2008
Gn1582Dr AM503366 Bousalem et al., 2009
Gn1583Dr AM503367 Bousalem et al., 2009
Bf103aDr AM503362 Bousalem et al., 2009
Gn158Dr AM503368 Bousalem et al., 2009
DpBV/K10 SB15b_DP AM072695 Kenyon et al., 2008
DeBV-E/K11 FJ75b De AM072662 Kenyon et al., 2008
PG176 De AM072686 Kenyon et al., 2008
K12 SB10aDn AM072692 Kenyon et al., 2008
WS31aDn AM421696 Kenyon et al., 2008
K13 PG102cDa AM421690 Kenyon et al., 2008
DBV-A(B)/U12 GHL2a Dr AM072665 Bousalem et al., 2009
Gn1551Dr AM503380 Bousalem et al., 2009
Gn842Dr AM503382 Bousalem et al., 2009
Gn155Dr AM503383 Bousalem et al., 2009
Gn84Dr AM503385 Bousalem et al., 2009
Gn501Dr AM503387 Bousalem et al., 2009
Gn5031Dr AM503388 Bousalem et al., 2009
DBV-D Gn1632Dr AM503399 Bousalem et al., 2009
Gn1645Da AM503401 Bousalem et al., 2009
T13 DBRTV1-2RT KX008597 Bömer et al., 2016
DBRTV1-3RT KX008598 Bömer et al., 2016
DBRTV1 KX008596 Bömer et al., 2016
NGb1844Dr1 KX008591 Bömer et al., 2016
T14 DBRTV2 KX008599 Bömer et al., 2016
DBRTV2-3RT KX008601 Bömer et al., 2016
DBRTV2-4RT KY827393 Bömer et al., 2016
DBRTV2-2RT KX008600 Bömer et al., 2016
T15 NGb0310Da3 KX008584 Bömer et al., 2016
TG2Dr AM944580 Eni et al., 2008
T16 NG165De KY555561 Turaki et al., 2017
T17 NGb53Dr KY555548 Turaki et al., 2017
99
S2 Table. Acronyms, GenBank accession and virus names of sequences used for
phylogenetic analysis in Fig 3B.
Genus Virus species Acronym GenBank
accession
Badnavirus Banana streak GF virus BSGFV AY493509
Banana streak IM virus BSIMV HQ593112
Banana streak MY virus BSMYV AY805074
Banana streak OL virus BSOLV AJ002234
Banana streak UA virus BSUAV HQ593107
Banana streak UI virus BSUIV HQ593108
Banana streak UL virus BSULV HQ593109
Banana streak UM virus BSUMV HQ593110
Banana streak VN virus BSVNV AY750155
Bougainvillea chlorotic vein banding
virus
BsCVBV EU034539
Cacao swollen shoot virus CSSV NC_001574
Citrus yellow mosaic virus CYMV AF347695
Commelina yellow mottle virus ComYMV X52938
Dioscorea bacilliform AL virus 2 DBALV2 DBALV2
Dioscorea bacilliform AL virus 2 DBALV X94578,
X94580,
X94582,
X94575
Dioscorea bacilliform ES virus DBESV DBESV
Dioscorea bacilliform RT virus 1 DBRTV1 KX008574
Dioscorea bacilliform RT virus 2 DBRTV2 KX008577,
KY827393
Dioscorea bacilliform RT virus 3 DBRTV3 MF476845
Dioscorea bacilliform SN virus DBSNV DQ822073
Dioscorea bacilliform TR virus DBTRV KX430257
Fig badnavirus 1 FBV-1 JF411989
Gooseberry vein banding associated
virus
GVBaV JQ316114
Grapevine roditis leaf discoloration-
associated virus
GRLDaV HG940503
Grapevine vein-clearing virus GVCV JF301669
Pagoda yellow mosaic associated virus PYMAV KJ013302
Pineapple bacilliform comosus virus PBCOV GU121676
Piper yellow mottle virus PYMoV KC808712
Rubus yellow net virus RYNV KM078034
Sugarcane bacilliform Guadeloupe A
virus
SCBGAV FJ824813
Sugarcane bacilliform Guadeloupe D
virus
SCBGDV FJ439817
Sugarcane bacilliform IM virus SCBIMV AJ277091
100
Sugarcane bacilliform MO virus SCBMOV NC_008017
Sweet potato caulimo-like virus SPCV HQ694978
Sweet potato vein clearing virus SPVCV HQ694979
Taro bacilliform virus TaBV AF357836
Caulimovirus Atractylodes mild mottle virus AMMV KR080327
Carnation etched ring virus CERV X04658
Cauliflower mosaic virus CaMV V00141
Dahlia mosaic virus DaMV JX272320
Figwort mosaic virus FMV X06166
Horseradish latent virus HRLV JX429923
Lamium leaf distortion virus LLDV EU554423
Mirabilis mosaic virus MiMV AF454635
Soybean Putnam virus SPuV JQ926983
Strawberry vein banding virus SVBV X97304
Cassava vein mosaic virus CsVMV U59751
Petuvirus Petunia vein clearing virus PVCV U95208
Rosadnavirus Rose yellow vein virus RYVV JX028536
Solendovirus Tobacco vein clearing virus TVCV AF190123
Soymovirus Blueberry red ringspot virus BRRV AF404509
Cestrum yellow leaf curling virus CmYLCV AF364175
Peanut chlorotic streak virus PCSV U13988
Soybean chlorotic mottle virus SbCMV X15828
Tungrovirus Rice tungro bacilliform virus RTBV NC001914
Unassigned Dioscorea nummularia-associated virus DNUaV MG944237
101
Chapter 5
An improved degenerate-primed rolling circle amplification
and next-generation sequencing approach for the detection
and characterization of badnaviruses
Amit Sukal1,2, Dawit B Kidanemariam1, James Dale1, Robert M. Harding1 and
Anthony James1*
1 Centre for Tropical Crops and Biocommodities, Queensland University of
Technology, Brisbane, Queensland, Australia
2 Centre for Pacific Crops and Trees, Pacific Community, Suva, Fiji.
* Corresponding author:
E-mail address: [email protected] (APJ)
[Formatted for submission to Virology]
102
QUT Verified Signature
QUT Verified Signature
QUT Verified Signature
QUT Verified Signature
QUT Verified Signature
103
Abstract
The genus Badnavirus is characterized by members that are genetically and
serologically heterogeneous making their detection and characterization difficult. The
presence of integrated badnavirus-like sequence in some host species further
complicates diagnosis using PCR-based protocols. To circumvent these issues, we
have optimized various RCA protocols including random-primed RCA (RP-RCA),
primer-spiked random-primed RCA (primer-spiked RP-RCA), directed RCA (D-
RCA) and specific-primed RCA (SP-RCA). For all methods, amplification of
badnavirus genomes is greatly improved using incubation temperatures of 36°C
instead of 30°C. Using Dioscorea bacilliform AL virus (DBALV) as an example, we
showed that viral DNA amplified using the optimized D-RCA and SP-RCA protocols
contained more than 80-fold badnavirus Illumina MiSeq-generated reads than those
amplified using random primed-RCA (RP-RCA). The optimized RCA techniques
described here were used to successfully amplify badnaviruses infecting banana
(BSCAV, BSGFV, BSMYV, BSOLV), sugar cane (SCBIMV), taro (TaBV) and yam
(DBALV, DBALV2, DBESV and DBRTV2).
Keywords: RCA, RP-RCA, primer-spiked R-RCA, D-RCA, SP-RCA, yam,
Dioscorea spp., badnavirus, NGS
1. Introduction
The genus Badnavirus (family Caulimoviridae) consists of plant pararetroviruses
that infect a wide range of economically important crops and cause estimated global
economic crop losses ranging from 10-90% (Bhat et al., 2016). Badnaviruses possess
non-enveloped bacilliform-shaped virions with an approximate size of 30 nm x 120-
104
150 nm (Geering and Hull, 2012). The genome consists of a single molecule of
circular, double-stranded DNA of 7.2-9.2 kb, typically encoding three open reading
frames (ORFs) all on the (+) strand (Geering, 2014). Replication occurs via reverse
transcription of a greater-than-genome length RNA which subsequently serves as a
template both for the translation of viral proteins and for reverse transcription to
replicate the genome (Borah et al., 2013; Geering and Hull, 2012; Iskra-Caruana et al.,
2014). The genomes of some badnavirus species are integrated into their host plant
genomes, and these sequences are referred to as endogenous badnaviruses (Bhat et al.,
2016; Hohn et al., 2008; Staginnus et al., 2009). These integration events are assumed
to have occurred through illegitimate recombination (Holmes, 2011) and/or during
DNA break repair (Gayral et al., 2008) rather than an association with viral infection.
However, there are some instances where these integrated sequences have given rise
to systemic virus infection following recombination events post exposure to abiotic
stress such as in vitro tissue culture process (Côte et al., 2010; Dallot et al., 2001) and
interspecific crossing (Lheureux et al., 2003).
Badnaviruses have been reported from the tropical and temperate regions of
Africa, Asia, Europe, Oceania and the Americas. Most badnavirus species infect
tropical and subtropical crops such as banana, black pepper, citrus, cocoa, sugarcane,
sweet potato, taro and yam, with a few known to infect plants of the temperate regions
such as gooseberry, grape, red raspberry, and ornamental spiraea (reviewed in Bhat et
al., 2016). Members of the genus Badnavirus are genetically and serologically
heterogeneous, having relatively low nucleotide identities, even within the same
species, when compared with other virus genera (Borah et al., 2013; Geering et al.,
2000; Harper et al., 2005, 2004; Jaufeerally-Fakim et al., 2006; Kenyon et al., 2008;
Lockhart et al., 1993).
105
The high sequence and serological diversity, and heterogeneous nature of
badnaviruses, in addition to the presence of endogenous badnavirus sequences,
presents challenges for the characterization and detection of these viruses and for the
safe exchange of germplasm. Although antibodies have been prepared against several
badnaviruses for use in serological-based detection tests, they often lack the utility to
detect all virus isolates (Kenyon et al., 2008; Seal et al., 2014). Molecular tools, such
as PCR, real-time-PCR and loop-mediated isothermal assays have also been developed
for several badnaviruses (reviewed in Bhat et al., 2016). However, these are
constrained by the highly heterogeneous nature of badnaviruses as well as the presence
of integrated sequences in some host plant genomes. Rolling circle amplification
(RCA) is a method that utilizes phi29 polymerase, an enzyme which preferentially
amplifies circular DNA and has strong strand displacement and 3`-5` proofreading
abilities. These features result in high-fidelity amplification (Blanco et al., 1989;
Rockett et al., 2015) and, as a result, the technique has been exploited as a sequence-
independent (random primed or RP) amplification strategy to characterize several
groups of DNA-viruses infecting humans, animals and plants (Johne et al., 2009).
However, the sequence-independent nature of RP-RCA can result in off-target
amplification, with some studies in sweet potato (Paprotka et al., 2010) and sugar beet
(Homs et al., 2008) reporting amplification of non-viral host DNA, such as
mitochondrial DNA. Until 2011, RCA was primarily used to detect plant viruses with
small genomes (<3 kb) belonging to the families Geminiviridae and Nanoviridae
(Grigoras et al., 2009; Haible et al., 2006; Inoue-Nagata et al., 2004). James et al.
(2011a) showed that RCA could be used to detect plant viruses with larger genomes
such as those from the family Caulimoviridae. Further, they reported an increase in
the amplification of the target banana streak virus (BSV, genus Badnavirus) sequences
106
through the addition of BSV-specific primers in addition to the random hexamers
included in premixed commercial kits, such as the TempliPhi kit (GE Healthcare,
United Kingdom). The study also highlighted the utility of RCA for the differential
amplification of episomal badnavirus genomes compared to their integrated
counterparts. The optimized RCA protocol has been subsequently used for the
characterization of novel badnaviruses infecting banana (James et al., 2011b), fig
(Laney et al., 2012) and yam (Bömer et al., 2016; Sukal et al., 2017). When using the
premixed commercial RCA kit protocols to amplify badnaviruses from yam, however,
Bömer et al. (2016) reported non-specific amplification of DNA from both circular
and linear non-viral templates.
The use of premixed kit components, either with/without additional virus-specific
primers, has been the standard for most badnavirus RCA applications. However,
whereas the use of these kits for the detection of badnaviruses in banana has proven
highly successful, their use for the detection of badnaviruses from other crops such as
yams has been somewhat less successful (Bömer et al., 2016). Unfortunately, the
nature of premixed kits, such as those supplied with the TempliPhi kit, precludes
significant scope for optimization. In this study, we report the development of an
optimized RCA-based method by manipulating the individual components of the RCA
reaction and by the inclusion of improved badnavirus degenerate primers. The use of
this method significantly increases episomal badnavirus genome amplification
compared to commercial premixed kits and can be used for badnavirus detection and
characterization using both Sanger and next-generation sequencing (NGS).
107
2. Materials and Methods
2.1. Samples
Total nucleic acid (TNA) from Dioscorea esculenta leaf tissues infected with
Dioscorea bacilliform AL virus (DBALV) isolate VUT02_De (GenBank accession
MG948562) and Dioscorea bacilliform ES virus (DBESV) isolate FJ14 (GenBank
accession KY827394), D. alata leaf tissue infected with Dioscorea bacilliform AL
virus 2 (DBALV2) isolate PNG10 (GenBank accession KY827395) and Dioscorea
rotundata leaf tissue infected with Dioscorea bacilliform RT virus 2 (DBRTV2) isolate
SAM01 (GenBank accession KY827393) was obtained from the Centre for Pacific
Crops and Trees (CePaCT), Pacific Community (SPC) germplasm collection in Fiji.
Banana leaf tissue infected with isolates of banana streak CA virus (BSCAV), banana
streak GF virus (BSGFV), banana streak MY virus (BSMYV) and banana streak OL
virus (BSOLV), as well as sugarcane infected with sugar cane bacilliform IM virus
(SCBIMV) and taro infected with taro bacilliform virus (TaBV), was provided by the
Centre for Tropical Crops and Biocommodities (CTCB), Queensland University of
Technology (QUT). TNA was extracted using a CTAB-based method (Kleinow et al.,
2009) and the yield and quality were assessed using a NanoDrop spectrophotometer
(ThermoFisher Scientific, Australia). The concentration of purified TNA was adjusted
to ~500 ng/μl with sterile nuclease-free water (sterile NF-H2O) for RCA experiments.
2.2. Primer design
The complete genome sequences of 182 badnaviruses, representing 43 species, were
accessed from GenBank. For each genome, the ORF 1 and ORF 2 sequences, as well
as the ORF 3 conserved domains equivalent to the cauliflower mosaic virus (CaMV)
movement protein (L43-E243 of ORF 1 protein), coat protein (L261-N429 of ORF 4),
108
aspartic protease (K36-Q120 of ORF 5), reverse transcriptase (K273-G449 of ORF 5) and
ribonuclease H (I547-E673) (Geering and Hull, 2012) were identified. The ORF 1, ORF
2, or individual ORF 3 conserved domain sequences were separately aligned using the
CLUSTALW algorithm in MEGA7. Primers were designed from the consensus
sequence of each alignment using Geneious® v11.0.4 (http://www.geneious.com;
Kearse et al., 2012). The specificity of each primer was assessed using both Primer-
BLAST at NCBI and in silico in Geneious® using the 182 complete genome sequences.
To circumvent the DNA exonuclease activity of phi29 polymerase, the two terminal
3’ nucleotides of each primer were phosphorothioate modified. A total of twenty eight
degenerate badnavirus primers were synthesized (Table 1), in addition to
phosphorothioate modified Badna-MFP/MRP (Turaki, 2014) and BadnaFP/RP (Yang
et al., 2003) primers. Episomal DBV-free accession, DA/NGA01, was used as a
negative control for the RCA experiments. DA/NGA01 is a Nigerian accession that
was obtained from International Institute of Tropical Agriculture (IITA) by SPC-
CePaCT. This accession was tested free of episomal DBV at IITA using IC-PCR and
retested at SPC-CePaCT using IC-PCR and RCA.
2.3. Random-primed RCA (RP-RCA)
RP-RCA was done using the Illustra TempliPhi 100 Amplification Kit (GE
Healthcare) essentially as described by the manufacturer with some modifications.
Briefly, a 1 μl aliquot of TNA (~500 ng) was mixed with 5 μl of kit sample buffer and
incubated at 95°C for 3 min, cooled to 4°C and placed on ice. The denatured sample
solution was then combined with 5 μl of reaction buffer premixed with 0.2 μl of phi29
polymerase. RP-RCA was either carried out at 30°C (manufacturer’s
109
recommendation) or 36°C for 18 h followed by 65°C for 10 min to inactivate the
enzyme.
2.4. Primer-spiked random-primed RCA (primer-spiked RP-RCA)
Primer-spiked RP-RCA was essentially as described for RP-RCA with the
addition of 1 μl of a mixture of 32 degenerate badnavirus primers (Table 1) at a final
concentration of 0.4 μM of each primer to 5 μl of the sample buffer as previously
described by James et al. (2011a) with either 30°C or 36°C as the incubation
temperature.
2.5. Directed RCA (D-RCA)
The directed RCA (D-RCA) protocol was a modification of the published two-step
RCA used for amplification of low-copy number human polyomaviruses, which
consisted of an annealing step followed by an amplification step (Marincevic-Zuniga
et al., 2012; Rockett et al., 2015). For the annealing step, the 32 badnavirus-specific
primer mix (Table 1) at a final concentration of 0.4 μM of each primer was combined
with 1 × phi29 buffer (NEB, Australia) and 1 μl TNA in a final volume of 10 μl. The
mixture was denatured at 95°C for 3 min, cooled to 4°C and placed on ice. A second
mixture consisting of 2.5 μM of exonuclease-resistant random hexamers
(ThermoFisher Scientific, Australia), 1 × phi29 buffer, 2 ng/μl bovine serum albumin
(BSA), 4 mM DTT, 15 mM dNTPs, 5 U of phi29 polymerase (ThermoFisher
Scientific, Australia) and sterile NF-H2O to 10 μl was prepared and combined with the
denatured primer/template mixture. Reactions were incubated at 36°C for 18 h,
followed by enzyme inactivation at 65°C for 10 min.
110
Table 1 Sequences of primers used in primer-spiked RP-RCA, D-RCA and SP-RCA
protocols
Primer name Sequence (5’ to 3’) Reference
Badna-MFP CAARTMTCTATCCTYACCAAAGG Turaki (2014)
Badna-MRP AWTGCYTGNACTCCATGRG Turaki (2014)
BadnaFP CCAYTTRCAIACISCICCCCAICC Yang et al. (2003)
BadnaRP ATGCCITTYGGIITIAARAAYGCICC Yang et al. (2003)
Badna_RCA4 AYNADSAGRRTTKGYYTCHCC This study
Badna_RCA5 AANYCRRCRTTDGRDGTRTTKG This study
Badna_RCA18 ACHYYNTSRATGBTKRTANKYRAA This study
Badna_RCA23 GGBTCAAKRAYDARYATDGCYCC This study
Badna_RCA12 CARHTRRTHTANRTHATMCCDRA This study
Badna_RCA45 TAYGGNRYMAGRARRRDCHA This study
Badna_RCA64 TTYGAYYTRAARWSYGGHTT This study
Badna_RCA65 TCHATMSMHTGGACDGCHTT This study
Badna_RCA78 ADAYKCCWCCCCAWCC This study
Badna_RCA17 TTYRSDRAYTAYSMRG This study
Badna_RCA7 TMCCWGCWGARGTVYTVTA This study
Badna_RCA8 TWYATYCAYMTHGGWRTVHT This study
Badna_RCA11 ATGGARGTDGAYYTDWCHRAAGG This study
Badna_RCA13 ATGAYVACHATHVRRGAYTTCTA This study
Badna_RCA46 TAYAARGGHAARCCWCA This study
Badna_RCA47 AAACHCATGTNMGRRTWGWHAA This study
Badna_RCA62 TGGTVTTCAAYTAYAARMG This study
Badna_RCA66 TDTAYGAATGGYTDGTHAT This study
Badna_RCA67 THTTYCARAGRAARATGG This study
Badna_RCA71 TGGRYTDRTYCTHAGYCC This study
Badna_RCA77 TVRTMMTWGARACWGAYGGHT This study
Badna_RCA80 AAYTTBCCRCTKGCRTADGCRCA This study
Badna_RCA81 ACHATYGAYGCHGARAT This study
Badna_RCA82 TYAARATHTAYTAYYTKGA This study
Badna_RCA83 ATDGCYTGRCWRTCWGTTCTKA This study
Badna_RCA86 TTBCCDTYWATRTGYTC This study
Badna_RCA1 TGGTATCAGAGCWDDGT This study
Badna_RCA14 TCNGTYTGYTTYTCDATRAAYTT This study
111
2.6. Specific-primed RCA (SP-RCA)
SP-RCA was carried out essentially as described for D-RCA except that the random
hexamers used in the second master mix were substituted with the badnavirus-specific
primer mixture at a final concentration of 0.4 μM of each primer.
2.7. Optimization of D-RCA and SP-RCA
To determine the optimum incubation temperature, RCA was carried out using TNA
from DBALV-infected yam as template. Incubation temperatures ranging from 30-
40°C, in increments of 2°C, were assessed and all reactions used an 18 h incubation
time. To investigate the optimum incubation time, RCA was carried out as before using
incubation times of 4, 8, 12, 16 and 18 h at the optimum RCA temperatures. To
determine the optimal dNTP concentration for amplification, RCA was carried out
using final concentrations of 0, 2.5, 5, 10, 15 and 20 mM dNTPs. The sensitivity of
RP-RCA, primer-spiked RP-RCA, D-RCA and SP-RCA was determined by varying
the template concentration using 500, 250, 125, 50, 25, 12.5 and 0 ng of DBALV-
infected yam TNA with RCA carried out using the optimized temperature (36°C),
incubation time and concentration of dNTPs. All RCA conditions were kept essentially
as described in sections 2.3 to 2.6 while varying the parameter under investigation.
2.8. Restriction analysis, cloning and Sanger sequencing
RCA products were independently digested with either EcoRI, KpnI, SphI or StuI,
which were selected based on in silico restriction analysis of published badnavirus
genome sequences, or from experimental experience, to generate useful restriction
profiles. RCA products (10 l) were digested in a total reaction volume of 20 l
containing 5 U of enzyme, 1 × CutSmart® buffer (NEB) and sterile NF-H2O. Reaction
112
mixtures were incubated at 37°C for 2-4 h and the digested RCA products were
analysed by electrophoresis through 1.5% agarose gels stained with SYBR® Safe
(ThermoFisher Scientific). Fragments of interest were purified using Freeze ‘N
SqueezeTM DNA Gel Extraction Spin Columns (Bio-Rad, Australia) and cloned into
pUC19 as described in Sukal et al. (2017). Sequencing was carried out using either
M13F/R or BadnaFP/RP primers.
2.9. RCA-NGS and genome assembly
To characterize the specificity of each of the different RCA protocols for amplification
of badnaviruses, DBALV TNA amplified using RP-RCA, D-RCA and SP-RCA was
purified and sequenced using the Illumina MiSeq platform. RCA products were
purified using the Illustra™ GFX™ PCR DNA and Gel Band Purification Kit (GE
Healthcare). Sequencing libraries were prepared from purified RCA products using
the Nextera™ XT Library Prep Kit (Illumina) and paired-end reads generated using
the MiSeq system (Illumina) at the Central Analytical Research Facility (CARF),
Queensland University of Technology, Brisbane, Australia. Raw read quality was
assessed with FastQC v0.10.1
(http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), with residual adapter
sequences trimmed, as well as low quality and short reads (<40 nt) removed, using the
BBduk plugin in Geneious®. To determine NGS read identities, the quality corrected
reads were mapped against badnavirus genomes (182 complete sequences), the
Dioscorea rotundata reference genome (GenBank accessions DF933857-DF938579),
plastid (GenBank accessions EF380353, KJ490011, KY085893) or mitochondrial
(GenBank accession LC219374) DNA using the Geneious reference mapper
algorithm. Unmapped reads were de novo assembled using SPAdes v3.5 (Bankevich
113
et al., 2012) and BLASTn was carried out on contigs >1000 bp to further determine
read identities. Finally, complete viral genomes were generated by reference mapping
total NGS reads against the DBALV genome (GenBank accession KX008571).
3. Results
3.1. Badnavirus RCA optimization
RCA optimization was initially carried out using TNA extracted from D.
esculenta originating from Vanuatu infected with DBALV isolate VUT02_De. The
effect of incubation temperature on RP-RCA, D-RCA and SP-RCA was evaluated
between 30 and 40°C at intervals of 2°C. Following RCA, a 10 μl aliquot of each
amplification reaction was digested with EcoRI, to generate four fragments of ~3.3,
1.7, 1.4 and 1 kb. Using RP-RCA, only very low levels of visible digest products were
observed irrespective of the RCA incubation temperatures (Fig. 1A). In contrast,
whereas incubation temperatures of 30 and 40°C also resulted in very poor
amplification of virus DNA using D-RCA and SP-RCA, considerably stronger visible
digest products were observed using incubation temperatures of 32-36°C for SP-RCA,
or 32-38°C for D-RCA (Fig. 1A). Similar results were observed when RCA reactions
were digested with SphI (results not shown).
The effect of dNTP concentration on amplification was evaluated by varying the
final concentration of dNTPs between 0 and 20 mM in the RCA reactions. When D-
RCA and SP-RCA-amplified DNA was digested with EcoRI, no visible reaction
products were observed in reactions with 0 or 2.5 mM dNTPs (Fig. 1B). In contrast,
visibly stronger digest products were observed using D-RCA reactions containing 5
mM dNTPs, while no visible digest products were observed from SP-RCA reactions
using 5 mM dNTPs. The strongest visible digest products were observed using D-RCA
114
and SP-RCA reactions containing dNTP concentrations in the range of 10-20 mM (Fig.
1B).
Evaluation of reaction incubation times showed that detectable levels of
badnavirus DNA were amplified after 12 h in both the D-RCA and SP-RCA reactions,
but only after 16 h from RP-RCA reactions (Fig. 1C). However, in all three RCA
protocols, optimal levels of visible digest products were observed following
incubation for 16-18 h.
When the template concentration was varied from 5 to 500 ng, very low levels of
visible digest products were observed using the RP-RCA protocol containing 250-500
ng of TNA template (Fig 2A). In contrast, digestion of products from primer-spiked
RP-RCA generated visible digest products when as little as 50 ng of TNA template
was used (Fig. 2B). The levels of visible digest products from both the RP-RCA and
primer-spiked RP-RCA protocol was positively correlated with the amount of TNA
template added, with higher starting template concentrations resulting in higher
amounts of visible digest products. When the amount of TNA template was varied in
the D-RCA and SP-RCA protocols, relatively high amounts of visible digest products
were observed at all TNA concentrations assessed (Fig. 2C and D, respectively).
Following optimization, D-RCA and SP-RCA were carried out as described in 2.5
and 2.6, respectively, with a final concentration of 15 mM dNTPs and an incubation
temperature of 36°C for a duration of 18 h, while, RP-RCA and primer-spiked RP-
RCA were carried out as described in 2.3 and 2.4, respectively. All RCA was carried
out with using 500 ng of TNA as template.
115
A
B
M
RP-RCA (°C) D-RCA (°C) SP-RCA (°C)
M30 32 34 36 38 40 30 32 34 36 38 40 30 32 34 36 38 40
10 kb
6 kb
3 kb
1 kb
GradientRCA
M
D-RCA (mM)
M
SP-RCA (mM)
M0 2.5 5 10 15 20 0 2.5 5 10 15 20
10 kb
6 kb
3 kb
1 kb
dNTPconcentration
116
C
Fig. 1. RCA of DBALV isolate VUT02_De. (A) Gradient incubation temperature from
30 to 40C in 2C increments for RP-RCA, D-RCA and SP-RCA, (B) D-RCA and SP-
RCA setup with dNTP concentrations of 0 to 20 mM, (C) Effect of incubation duration
on RP-RCA, D-RCA and SP-RCA. The RCA products in (A) and (B) were digested
with EcoRI, while, RCA products of (C) were digested with SphI and electrophoresed
through 1.5% agarose gel stained with SYBR® Safe. M - GeneRuler 1 kb DNA Ladder
(ThermoFisher Scientific, Australia) visible are lanes from 1 kb onwards. The
numbers on the side indicate the molecular sizes of the markers in base pairs.
117
Fig. 2. RCA of DBALV isolate VUT02_De using concentrations of 0 to 500 ng total nucleic acid. (A) RP-RCA, (B) primer-spiked RP-RCA (C)
D-RCA and (D) SP-RCA. The RCA products were digested with EcoRI and electrophoresed through 1.5% agarose gels stained with SYBR®
Safe. NT- No template control. M - GeneRuler 1 kb DNA Ladder (ThermoFisher Scientific, Australia).
118
3.2. RP-RCA, primer-spiked RP-RCA, D-RCA and SP-RCA amplification of
badnaviruses
To compare the utility of the optimized RCA protocol with previously described
RCA protocols for the detection of a broad range of badnaviruses, TNA from bananas
infected with BSCAV, BSGFV, BSMYV or BSOLV, yam plants infected with
DBALV, DBALV2, DBESV or DBRTV2, sugar cane infected with SCBIMV and taro
infected with TaBV were subjected to RP-RCA, primer-spiked RP-RCA, D-RCA and
SP-RCA using the optimized reaction conditions. Since temperatures in the range of
34-38°C were shown to increase the efficiency of both RP-RCA (Fig. 1A) and primer-
spiked RP-RCA (figure not shown), the performance of RP-RCA and primer-spiked
RP-RCA was evaluated at both 30°C (used in previous published studies), and at 36°C.
To obtain putative full-length restriction digestion fragments, RCA products from
TNA extracts containing BSCAV, BSGFV, BSMYV, BSOLV, DBESV and SCBIMV
were digested with KpnI, RCA products from TNA extracts containing DBALV,
DBALV2, DBRTV2 were digested with SphI while RCA products from the TNA
extract containing TaBV was digested with StuI. RP-RCA carried out at 30°C
amplified large amounts of BSGFV and BSMYV (Fig. 3A, lanes 2-3), however, only
very low levels of amplification were observed for BSCAV, DBALV2, SCBIMV and
TaBV (Fig. 3A, lanes 1, 6 and 9-10) and no visible digest products were present in
digests of RCA-amplified DNA from samples with BSOLV, DBALV, DBALV2 and
DBRTV2 (Fig. 3A, lanes 4-5, 7-8). In contrast, RP-RCA carried out at 36°C amplified
all badnavirus species tested, with highest levels of amplification observed with
BSGFV, BSMYV, DBALV, DBALV2 and SCIMV (Fig. 3B, lane 2-3 5-6 and 9)
compared to BSCAV, BSOLV, DBESV, DBRTV2 and TaBV (Fig. 3B, lane 1, 4, 7-8
and 10).
119
Viral DNA was amplified from all samples using primer-spiked RP-RCA at 30°C,
although the amplification products from some samples containing BSCAV, BSGFV,
BSMYV and SCIMV (Fig. 3C, lanes 1-3 and 9) were comparatively higher than others
(BSOLV, DBALV, DBALV2, DBESV, DBRTV2 and TaBV; Fig. 3C lanes 4-8 and
10). The use of primer-spiked RP-RCA at 36°C also resulted in the amplification of
all viruses, with either similar intensity reaction products (eg. BSCAV) or relatively
stronger amplification products observed, compared with incubation at 30°C.
Comparatively lower amplification was observed for all badnaviruses from yam host
with primer-spiked RP-RCA at 30°C (Fig. 3C, lanes 5-8) compared with 36°C (Fig.
3D, lanes 5-8).
D-RCA and SP-RCA amplified all samples (Fig. 3E and F, respectively), although
consistently higher amplification was achieved for all samples with D-RCA compared
to RP-RCA, primer-spiked RP-RCA and SP-RCA. Whereas consistently high levels
of amplification were obtained using D-RCA (Fig. 3E), the level of amplification from
other RCA protocols was variable.
All the single digest bands of RP-RCA (30 and 36°C), primer-spiked RP-RCA
(30 and 36°C) D-RCA and SP-RCA (Fig. E and F) were excised, cloned into
appropriately cut pUC19 and Sanger sequenced using M13F/R or BadnaFP/RP
primers. Sequencing confirmed that the amplification was of the respective virus
isolates used for the RCA optimization.
120
121
Fig. 3. Different badnavirus infected samples amplified with (A) RP-RCA at 30°C incubation, (B) RP-RCA at 36°C incubation, (C) primer-spiked
RP-RCA at 30°C, (D) primer-spiked RP-RCA at 36°C, (E) D-RCA and (F) SP-RCA. Lanes 1-4, 7 and 9 represent KpnI digested RCA products of
BSCAV, BSGFV, BSMYV, BSOLV, DBESV and SCIMV, respectively, while lanes 5-6 and 8 represents SphI digested RCA of DBALV,
DBALV2, DBRTV2 and lane 10 represents TaBV digested with StuI. Lane 11 is a known negative sample (DA/NGA01) digested with EcoRI and
Lane 12 is a no template control. M - GeneRuler 1 kb DNA Ladder (ThermoFisher Scientific, Australia) visible are lanes from 1 kb onward.
122
3.3. RCA-NGS for virus characterization
To investigate the efficiency of the different RCA protocols for the amplification
of badnavirus DNA, TNA extracted from DBALV-infected D. esculenta from
Vanuatu (DBALV isolate VUT02_De) was used in RP-RCA, D-RCA and SP-RCA.
Undigested reaction products of single RP-RCA, D-RCA and SP-RCA reactions were
then individually sequenced using the Illumina platform which resulted in paired-end
reads of 350,272, 604,382 and 800,560 from NGS, respectively. Following adapter
removal, quality trimming and removal of short reads (<40 nt) from the paired-end
reads, 317,608, 557,088 and 751,680 respective reads from the RP-RCA, D-RCA and
SP-RCA products were obtained. Reference mapping using Geneious revealed that
0.15%, 2.39%, 41.85%, and 1.38% of RP-RCA sequences, 85.71%, 4.00%, 0.41% and
0.03% of D-RCA sequences and 84.78%, 2.96%, 0.30% and 0.02% of SP-RCA
sequences mapped to badnaviruses, the Dioscorea rotundata reference genome
sequence, plastid or mitochondrial sequences, respectively, while 54.27%, 9.86% and
11.94% of RP-RCA, D-RCA and SP-RCA generated sequences remained unmapped.
When the unmapped reads were de novo assembled and contigs >1000 bp were
subjected to BLASTn analysis, the majority of hits were to plant genomes other than
Dioscorea spp.
Reference mapping was further carried out in Geneious, using DBALV-[2ALa]
(GenBank accession KX008571) as a reference, to generate the complete genome of
isolate VUT02_De used in this study. The Geneious mapper assigned 7,600 (mean
coverage 11), 477,485 (mean coverage 12,783) and 637,254 reads (mean coverage
17,145) of the trimmed reads generated using RP-RCA, D-RCA and SP-RCA,
respectively, to DBALV-[2ALa]. The D-RCA and SP-RCA libraries generated a
123
complete circular virus genome using the NGS data, whereas the RP-RCA library only
generated fragmented sequences, the largest of which was 7,203 nt.
3.4. DBALV isolate VUT02_De genome
The complete NGS-derived sequences of DBALV isolate VUT02_De generated
using D-RCA and SP-RCA NGS showed 99.6% nucleotide identity to each other. The
consensus genome of DBALV isolate VUT02_De comprised 7,509 nt and contained
three open reading frames (ORFs). ORF 1 comprised 432 bp and encoded a putative
protein of 144 amino acids (aa), while ORF 2 was 378 bp and encoded a putative
protein of 126 aa. ORF 3 was 5,682 bp and encoded a putative protein of 1,894 aa. An
intergenic region (IR) of 1,022 bp was present between ORF 1 and ORF 3 and
contained a putative plant tRNAmet binding site (3`-TGGTATCAGAGCTTGGTT-5`1-
18) complementary to the consensus sequence of plant cytoplasmic initiator tRNAmet
(3`-ACCAUAGUCUCGGUCCAA-5′) which was designated as the start of the
circular viral genome. The 529 bp partial RT/RNase H-coding region delineated by
the BadnaFP/RP primers showed highest nucleotide identity (99.6%) to a partial
RT/RNase H-coding sequence of Dioscorea bacilliform virus isolate VU249_Db
(GenBank accession AM072705) and 92.5-94.5% similarity to two other partial
sequences, VU254_DP and VU252_Db (AM072707 and AM072706, respectively)
originating from Vanuatu. When compared to published DBALV full length
sequences, the complete genome sequence DBALV-VUT02_DE had highest sequence
identities with DBALV isolate DBALV-[2ALa] (89.8%, KX008572), DBALV-[2Alb]
(89.7%, KX008571) and DBALV-[3RT] (85.5%, KX008595). The complete genome
sequence of DBALV-VUT02_DE has been deposited in GenBank under accession
number MG948562.
124
4. Discussion
In this paper, two improved RCA protocols for the amplification and
characterization of badnaviruses are described. The major advantage of these protocols
over previously described methods is that they avoid the use of premixed reaction
components, such as those included in the commercial TempliPhi kit, thereby allowing
each component of the reaction mixture to be varied to achieve optimum viral genome
amplification. The use of a suite of badnavirus degenerate primers in the denaturation
step of these optimized protocols significantly increased the amplification bias towards
the target virus thus reducing non-target amplification.
Although RCA using kit-based protocols, such as TempliPhi, has been
successfully used to amplify badnaviruses from various host species, such as banana
(Baranwal et al., 2014; Carnelossi et al., 2014; James et al., 2011b; Javer-Higginson
et al., 2014; Sharma et al., 2015, 2014; Wambulwa et al., 2013, 2012), cacao
(Chingandu et al., 2017a, 2017b; Muller et al., 2018), fig (Laney et al., 2012),
mulberry (Chiumenti et al., 2016), Rubus spp. (Diaz-Lara et al., 2015) and yam (Bömer
et al., 2018, 2016; Sukal et al., 2017; Umber et al., 2014), the method still has several
limitations. Due to the sequence-independent nature of the kit-based RCA protocols,
plant-genome derived DNAs, such as mitochondrial or chloroplast DNA, are
sometimes also amplified (Bömer et al., 2016; Homs et al., 2008; Paprotka et al.,
2010). Further, during the initial annealing process, the random hexamer primers in
the premixed sample buffer bind to all available nucleic acids and not just the preferred
target sequences, enabling non-target DNA amplification in addition to the desired
target. Although the addition of virus-specific primers reported by James et al (2011a)
creates a bias towards the target badnavirus genomes, non-specific amplification still
occurs as a consequence of priming to non-target DNA by the random primers present
125
in the premixed sample buffer used in the denaturation step. Modification of the initial
denaturation mixture to only contain primers which anneal to the target virus
sequences results in amplification which is biased towards the target circular virus
genomes. Compared to random-primed RCA using the TempliPhi kit, where <1% of
the NGS reads mapped to badnavirus sequences, the use of either D-RCA or SP-RCA
resulted in 85.71% and 84.78% of respective reads mapping to the target badnavirus,
showing that these RCA protocols greatly enhance amplification of the target virus
genome.
In an effort to optimize the RCA protocol to detect the greatest breadth of
badnavirus sequence diversity, in silico primer-binding analysis using the 32
degenerate primers developed in this study was carried out using complete genome
sequences of the 43 currently recognized badnavirus species. This analysis showed
that at least 10 of the primers were able to bind to every complete genome sequence.
Using these primers, the SP- and D-RCA protocols were shown to successfully
amplify distantly related badnaviruses including four distinct BSVs, four distinct
DBVs, as well as SCBIMV and TaBV, from four different host plant species,
highlighting the utility of the method for both detection and characterization of
badnaviruses.
We also found that increasing incubation temperature to 36°C greatly improved
the performance of kit-based RCA protocols such as the RP-RCA and primer-spiked
RP-RCA (Fig. 3B and D, respectively). However, D-RCA was found to be the most
consistent and reproducible protocol for generic badnavirus amplification from the
different host plants. Although SP-RCA performed to an equivalent level in some
cases, there was some variability in amplification levels possibly due to variability in
the number of primers binding to different target virus genomes. However, the
126
reliability of SP-RCA should be improved further by designing specific primers for
the target virus species of interest.
RCA post-amplification analysis often involves restriction analysis to confirm
viral genome amplification, however, this is dependent on knowledge of suitable
restriction enzymes which generate reproducible restriction profiles. The genomic
heterogeneity of many badnaviruses, together with the limited availability of complete
genome sequences for some virus species, complicates the use of restriction analysis
for virus detection. By utilizing NGS of total undigested RCA products, both virus
detection as well as full genome characterization can be accomplished. The
combination of RCA and NGS has previously been used for the characterization of
circular DNA viruses including geminiviruses (Leke et al., 2016; Zubair et al., 2017;
Idris et al., 2014; Kathurima et al., 2016) and badnaviruses (Chingandu et al., 2017a,
2017b; Muller et al., 2018). Previous work using RP-RCA-NGS to characterize the
badnaviruses, Cacao mild mosaic virus (CaMMV) and Cacao yellow vein-banding
virus (CYVBV), showed that of the total 2,111,947 and 3,664,739 NGS reads obtained
by sequencing of RCA products, only 1,084,938 and 15,355 reads (representing
51.37% and 0.4% of the total reads, respectively) were derived from the target viral
sequences (Chingandu et al., 2017b). Using the SP- and D-RCA methods described
herein produced a far greater percentage of reads (~85%) mapping to the target
DBALV2 genome compared with the RP-RCA protocol (~1%). This result highlights
the significance of the improvement in target sequence amplification by omitting
random primers from the initial denaturation/annealing step as well as the utility for
using NGS to diagnose and characterize badnavirus genomes. The costs associated
with NGS may preclude its use as a routine diagnostic tool, however, if enough
127
badnavirus genome information can be amassed through initial RCA-NGS efforts,
RCA restriction analysis can then be used as an effective diagnostic tool.
The high levels of heterogeneity at both serological and genetic levels and the
presence of host genome integrated sequences believed to be remnants of ancient viral
sequences make characterization and diagnosis of some badnaviruses difficult. The
optimized RCA protocols described in this study coupled with NGS can be used for
the characterization and detection of badnaviruses from a range of host species. The
potential for using restriction analysis of either D-RCA or SP-RCA products as a
diagnostic tool for badnavirus detection remains high, with continual sequencing of
complete genomes improving knowledge of suitable restriction enzymes for digestion
of reaction products, particularly for the host/badnavirus combinations described in
this study.
Acknowledgements
The authors would like to thank the Centre for Pacific Crops and Trees (CePaCT) of
the Pacific Community (SPC) for making their yam collections available for this
project. Authors would also like to thank Dr. Michael Furlong and Dr. Grahame
Jackson for their support and advice on this research. This work was funded under the
Australian Centre for International Agricultural Research project PC/2010/065. AS is
a John Allwright Fellowship recipient.
128
References
Bankevich, A., Nurk, S., Antipov, D., Gurevich, A.A., Dvorkin, M., Kulikov, A.S.,
Lesin, V.M., Nikolenko, S.I., Pham, S., Prjibelski, A.D., Pyshkin, A. V., Sirotkin,
A. V., Vyahhi, N., Tesler, G., Alekseyev, M.A., Pevzner, P.A., 2012. SPAdes: A
new genome assembly algorithm and its applications to single-cell sequencing. J.
Comput. Biol. 19, 455–477.
Baranwal, V.K., Sharma, S.K., Khurana, D., Verma, R., 2014. Sequence analysis of
shorter than genome length episomal Banana streak OL virus like sequences
isolated from banana in India. Virus Genes 48, 120–127.
Bhat, A., Hohn, T., Selvarajan, R., 2016. Badnaviruses: The current global scenario.
Viruses 8, 177.
Blanco, L., Bernads, A., Lharo, J.M., Martins, G., Garmendia, C., 1989. Highly
efficient DNA synthesis by the phage 429 DNA polymerase. The J. Biol. Chem.
264, 8935-8940.
Bömer, M., Rathnayake, A.I., Visendi, P., Silva, G., Seal, S.E., 2018. Complete
genome sequence of a new member of the genus Badnavirus, Dioscorea
bacilliform RT virus 3, reveals the first evidence of recombination in yam
badnaviruses. Arch. Virol. 163, 533–538.
Bömer, M., Turaki, A., Silva, G., Kumar, P., Seal, S., 2016. A sequence-independent
strategy for amplification and characterization of episomal badnavirus sequences
reveals three previously uncharacterized yam badnaviruses. Viruses 8, 188.
Borah, B.K., Sharma, S., Kant, R., Johnson, A.M.A.A., Saigopal, D.V.R., Dasgupta,
I., 2013. Bacilliform DNA-containing plant viruses in the tropics: Commonalities
within a genetically diverse group. Mol. Plant Pathol. 14, 759–771.
Carnelossi, P.R., Bijora, T., Facco, C.U., Silva, J.M., Picoli, M.H.S., Souto, E.R.,
129
Oliveira, F.T. De, 2014. Episomal detection of banana streak OL virus in single
and mixed infection with Cucumber mosaic virus in banana “Nanicão Jangada.”
Trop. Plant Pathol. 39, 342–346.
Chingandu, N., Kouakou, K., Aka, R., Ameyaw, G., Gutierrez, O.A., Herrmann, H.-
W., Brown, J.K., 2017a. The proposed new species, cacao red vein virus, and
three previously recognized badnavirus species are associated with cacao swollen
shoot disease. Virol. J. 14, 199.
Chingandu, N., Zia-ur-rehman, M., Sreenivasan, T.N., Surujdeo-Maharaj, S.,
Umaharan, P., Gutierrez, O.A., Brown, J.K., Thyail, M.Z., Zia-ur-rehman, M.,
Sreenivasan, T.N., Surujdeo-Maharaj, S., Umaharan, P., Gutierrez, O.A., Brown,
J.K., Thyail, M.Z., 2017b. Molecular characterization of previously elusive
badnaviruses associated with symptomatic cacao in the New World. Arch. Virol.
162, 1363–1371.
Chiumenti, M., Morelli, M., De Stradis, A., Elbeaino, T., Stavolone, L., Minafra, A.,
2016. Unusual genomic features of a badnavirus infecting mulberry. J. Gen.
Virol. 97, 3073–3087.
Côte, F.X., Galzi, S., Folliot, M., Lamagnère, Y., Teycheney, P., 2010.
Micropropagation by tissue culture triggers differential expression of infectious
endogenous Banana streak virus sequences (eBSV) present in the B genome of
natural and synthetic interspecific banana plantains. Mol. Plant Pathol. 11, 137–
144.
Dallot, S., Acuña, P., Rivera, C., Ramírez, P., Cote, F., Lockhart, B.E.L., Caruana,
M.L., 2001. Evidence that the proliferation stage of micropropagation procedure
is determinant in the expression of Banana streak virus integrated into the genome
of the FHIA 21 hybrid (Musa AAAB). Arch. Virol. 146, 2179–2190.
130
Diaz-Lara, A., Mosier, N.J., Keller, K.E., Martin, R.R., 2015. A variant of Rubus
yellow net virus with altered genomic organization. Virus Genes 50, 104–110.
Gayral, P., Noa-Carrazana, J.-C., Lescot, M., Lheureux, F., Lockhart, B.E.L.,
Matsumoto, T., Piffanelli, P., Iskra-Caruana, M.-L., 2008. A single banana streak
virus integration event in the banana genome as the origin of infectious
endogenous pararetrovirus. J. Virol. 82, 6697–6710.
Geering, A.D., 2014. Caulimoviridae (Plant Pararetroviruses), in: ELS. John Wiley &
Sons, Ltd, Chichester, UK.
Geering, A.D., McMichael, L.A., Dietzgen, R.G., Thomas, J.E., 2000. Genetic
diversity among banana streak virus isolates from Australia. Phytopathology 90,
921–927.
Geering, A.D.W., Hull, R., 2012. Family Caulimoviridae, in: King, A.M.Q., Adams,
M.J., Carstens, E.B., Lefkowitz, E.J. (Eds.), Virus Taxonomy. Ninth Report of
the International Committee on Taxonomy of Viruses. Elsevier Academic Press,
Amsterdam, The Netherlands, pp. 429–443.
Grigoras, I., Timchenko, T., Katul, L., Grande-Pérez, A., Vetten, H.-J., Gronenborn,
B., 2009. Reconstitution of authentic nanovirus from multiple cloned DNAs. J.
Virol. 83, 10778–10787.
Haible, D., Kober, S., Jeske, H., 2006. Rolling circle amplification revolutionizes
diagnosis and genomics of geminiviruses. J. Virol. Methods 135, 9–16.
Harper, G., Hart, D., Moult, S., Hull, R., Geering, A., Thomas, J., 2005. The diversity
of banana streak virus isolates in Uganda. Arch. Virol. 150, 2407–2420.
Harper, G., Hart, D., Moult, S., Hull, R., 2004. Banana streak virus is very diverse in
Uganda. Virus Res. 100, 51–56.
Hohn, T., Richert-Pöggeler, K.R., Staginnus, C., Harper, G., Schwarzacher, T., Teo,
131
C.H., Teycheney, P.Y., Iskra-Caruana, M.L., Hull, R., 2008. Evolution of
integrated plant viruses. Plant Virus Evol. 53–81.
Holmes, E.C., 2011. The evolution of endogenous viral elements. Cell Host Microbe
10, 368–377.
Homs, M., Kober, S., Kepp, G., Jeske, H., 2008. Mitochondrial plasmids of sugar beet
amplified via rolling circle method detected during curtovirus screening. Virus
Res. 136, 124–129.
Inoue-Nagata, A.K., Albuquerque, L.C., Rocha, W.B., Nagata, T., 2004. A simple
method for cloning the complete begomovirus genome using the bacteriophage
φ29 DNA polymerase. J. Virol. Methods 116, 209–211.
Idris, A., Al-saleh, M., Piatek, M.J., Al-shahwan, I., Ali, S., Brown, J.K., Marek, J.,
Judith, K., Idris, A., Piatek, M.J., Ali, S., Al-saleh, M., Piatek, M.J., Al-shahwan,
I., Ali, S., Brown, J.K., 2014. Viral metagenomics: Analysis of begomoviruses
by illumina high-throughput sequencing. Viruses 6, 1219–1236.
Iskra-Caruana, M. line, Duroy, P.O., Chabannes, M., Muller, E., 2014. The common
evolutionary history of badnaviruses and banana. Infect. Genet. Evol. 21, 83–89.
James, A.P., Geijskes, R.J., Dale, J.L., A., Harding, R.M., 2011a. Development of a
novel rolling-circle amplification technique to detect banana streak virus that also
discriminates between integrated and episomal virus sequences. Plant Dis. 95,
57–62.
James, A.P., Geijskes, R.J., Dale, J.L., Harding, R.M., 2011b. Molecular
characterization of six badnavirus species associated with leaf streak disease of
banana in East Africa. Ann. Appl. Biol. 158, 346–353.
Jaufeerally-Fakim, Y., Khorugdharry, A., Harper, G., 2006. Genetic variants of banana
streak virus in Mauritius. Virus Res. 115, 91–98.
132
Javer-Higginson, E., Acina-Mambole, I., González, J.E., Font, C., González, G.,
Echemendía, A.L., Muller, E., Teycheney, P.Y., 2014. Occurrence, prevalence
and molecular diversity of banana streak viruses in Cuba. Eur. J. Plant Pathol.
138, 157–166.
Johne, R., Müller, H., Rector, A., van Ranst, M., Stevens, H., 2009. Rolling-circle
amplification of viral DNA genomes using phi29 polymerase. Trends Microbiol.
17, 205–211.
Kathurima, T.M., Ateka, E.M., Nyende, A.B., Holton, T., 2016. The rolling circle
amplification and next generation sequencing approaches reveal genome wide
diversity of Kenyan cassava mosaic geminivirus. African J. Biotechnol. 15,
2045–2052.
Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S., Buxton,
S., Cooper, A., Markowitz, S., Duran, C., Thierer, T., Ashton, B., Meintjes, P.,
Drummond, A., 2012. Geneious Basic: An integrated and extendable desktop
software platform for the organization and analysis of sequence data.
Bioinformatics 28, 1647–1649.
Kenyon, L., Lebas, B.S.M., Seal, S.E., 2008. Yams (Dioscorea spp.) from the South
Pacific Islands contain many novel badnaviruses: implications for international
movement of yam germplasm. Arch. Virol. 153, 877–889.
Kleinow, T., Nischang, M., Beck, A., Kratzer, U., Tanwir, F., Preiss, W., Kepp, G.,
Jeske, H., 2009. Three C-terminal phosphorylation sites in the Abutilon mosaic
virus movement protein affect symptom development and viral DNA
accumulation. Virol. 390, 89–101.
Laney, A.G., Hassan, M., Tzanetakis, I.E., 2012. An integrated badnavirus is prevalent
in fig germplasm. Phytopathology 102, 1182–9.
133
Leke, W.N., Khatabi, B., Mignouna, D.B., Brown, J.K., Fondong, V.N., 2016.
Complete genome sequence of a new bipartite begomovirus infecting cotton in
the Republic of Benin in West Africa. Arch. Virol. 161, 2329–2333.
Lheureux, F., Carreel, F., Jenny, C., Lockhart, B.E.L., Iskra-Caruana, M.L., 2003.
Identification of genetic markers linked to banana streak disease expression in
inter-specific Musa hybrids. Theor. Appl. Genet. 106, 594–598.
Lockhart, B.E.L., Olszewski, N.E., Bouhida, M., Lockhartz, B.E.L., Olszewski, N.E.,
Lockhart, B.E.L., Olszewski, N.E., Bouhida, M., Lockhartz, B.E.L., Olszewski,
N.E., 1993. An analysis of the complete sequence of a sugarcane bacilliform virus
genome infectious to banana and rice. J. Gen. Virol. 74, 15–22.
Marincevic-zuniga, Y., Gustavsson, I., Gyllensten, U., 2012. Multiply-primed rolling
circle amplification of human papillomavirus using sequence-specific primers.
Virology 432, 57–62.
Muller, E., Ravel, S., Agret, C., Abrokwah, F., Dzahini-Obiatey, H., Galyuon, I.,
Kouakou, K., Jeyaseelan, E.C., Allainguillaume, J., Wetten, A., 2018. Next
generation sequencing elucidates cacao badnavirus diversity and reveals the
existence of more than ten viral species. Virus Res. 244, 235–251.
Paprotka, T., Boiteux, L.S., Fonseca, M.E.N., Resende, R.O., Jeske, H., Faria, J.C.,
Ribeiro, S.G., 2010. Genomic diversity of sweet potato geminiviruses in a
Brazilian germplasm bank. Virus Res. 149, 224–233.
Rockett, R., Barraclough, K.A., Isbel, N.M., Dudley, K.J., Nissen, M.D., Sloots, T.P.,
Bialasiewicz, S., 2015. Specific rolling circle amplification of low-copy human
polyomaviruses BKV, HPyV6, HPyV7, TSPyV, and STLPyV. J. Virol. Methods
215–216, 17–21.
Seal, S., Turaki, A., Muller, E., Kumar, P.L., Kenyon, L., Filloux, D., Galzi, S., Lopez-
134
Montes, A., Iskra-Caruana, M.L., 2014. The prevalence of badnaviruses in West
African yams (Dioscorea cayenensis-rotundata) and evidence of endogenous
pararetrovirus sequences in their genomes. Virus Res. 186, 144–154.
Sharma, S.K., Vignesh Kumar, P., Geetanjali, A.S., Pun, K.B., Baranwal, V.K., 2015.
Subpopulation level variation of banana streak viruses in India and common
evolution of banana and sugarcane badnaviruses. Virus Genes 50, 450–465.
Sharma, S.K., Vignesh Kumar, P., Poswal, R., Rai, R., Swapna Geetanjali, A., Prabha,
K., Jain, R.K., Baranwal, V.K., 2014. Occurrence and distribution of banana
streak disease and standardization of a reliable detection procedure for routine
indexing of banana streak viruses in India. Sci. Hortic. 179, 277–283.
Staginnus, C., Iskra-Caruana, M.L., Lockhart, B., Hohn, T., Richert-Pöggeler, K.R.,
2009. Suggestions for a nomenclature of endogenous pararetroviral sequences in
plants. Arch. Virol. 154, 1189–1193.
Sukal, A., Kidanemariam, D., Dale, J., James, A., 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 Res. 238,
29–34.
Turaki, A.A., 2014. Characterization of badnavirus Sequences in West African Yams
(Dioscorea spp.). PhD thesis, University of Greenwich, United Kingdom, 240.
Umber, M., Filloux, D., Muller, E., Laboureau, N., Galzi, S., Roumagnac, P., Iskra-
Caruana, M.-L., Pavis, C., Teycheney, P.-Y., Seal, S.E., 2014. The genome of
African yam (Dioscorea cayenensis-rotundata complex) hosts endogenous
sequences from four distinct badnavirus species. Mol. Plant Pathol. 15, 790–801.
Wambulwa, M.C., 2012. Rolling circle amplification is more sensitive than PCR and
serology-based methods in detection of banana streak virus in Musa germplasm.
135
Am. J. Plant Sci. 03, 1581–1587.
Wambulwa, M.C., Wachira, F.N., Karanja, L.S., Kiarie, S.M., Muturi, S.M., 2013.
The influence of host and pathogen genotypes on symptom severity in banana
streak disease. African J. Biotechnol. 12, 27–31.
Yang, I.C., Hafner, G.J., Dale, J.L., Harding, R.M., 2003. Genomic characterization
of taro bacilliform virus. Arch. Virol. 148, 937–949.
Zubair, M., Zaidi, S.S.-A., Shakir, S., Farooq, M., Amin, I., Scheffler, J.A., Scheffler,
B.E., Mansoor, S., 2017. Multiple begomoviruses found associated with cotton
leaf curl disease in Pakistan in early 1990 are back in cultivated cotton. Sci. Rep.
7, 680.
136
This page is intentionally left blank
137
Chapter 6
Characterization and genetic diversity of Dioscorea
bacilliform viruses infecting Pacific yam germplasm
collections
Amit C. Sukal1, 2 Dawit B. Kidanemariam1 James L. Dale1 Robert M. Harding1
Anthony P. James1*
1 Centre for Tropical Crops and Biocommodities, Queensland University of
Technology, Brisbane, Queensland, Australia
2 Centre for Pacific Crops and Trees, Pacific Community, Suva, Fiji.
* Corresponding author:
E-mail address: [email protected] (APJ)
[Formatted for submission to Plant Pathology]
138
QUT Verified Signature
QUT Verified Signature
QUT Verified Signature
QUT Verified Signature
QUT Verified Signature
139
Abstract
Dioscorea bacilliform viruses (DBVs) are members of the family Caulimoviridae,
genus Badnavirus and are important pathogens of yam (Dioscorea spp.). DBVs are
difficult to diagnose due to their genetic and serological diversity, and this is further
complicated by the fact that some DBV species occur as endogenous sequences. To
date, the complete genome sequences of eight virus species have been determined,
with an additional eight putative species described based on partial RT-RNase H-
coding sequences. Using RCA, we screened 224 accessions in a Pacific yam
germplasm collection and 35 tested positive, with sequencing confirming all positive
samples as either Dioscorea bacilliform AL virus (DBALV) or Dioscorea bacilliform
AL virus 2 (DBALV2). DBALV was only present in Vanuatu and Tonga, while
DBALV2 was restricted to Papua New Guinea. Twenty complete genome sequences
were generated, including 10 of DBALV with a nucleotide sequence identity ranging
from 89 to 90%, and 10 of DBALV2 with a nucleotide sequence identity ranging from
87 to 89%. Based on these results DBALV and DBALV2 appear to be the most
prevalent badnavirus species infecting yams in the Pacific region. Analysis of NGS
reads from RCA-negative samples failed to identify sequences with similarity to
badnaviruses, further demonstrating the potential of RCA for detecting DBVs. For
both DBALV and DBALV2, several distinct restriction profiles were observed
amongst isolates of each species providing support for the existence of sequence
variants. The direct sequencing of RCA products using NGS highlights the utility of
RCA for episomal virus characterization, which will lead to improved diagnostics to
support the safe exchange of yam germplasm.
Keywords: Yam, badnavirus, Dioscorea bacilliform virus, DBALV, DBALV2, D-
RCA, RCA-NGS
140
Introduction
Yams (Dioscorea spp.) provide a staple food source for millions of people in
Africa, South America, Asia and the Pacific. It is also a crop that has significant
cultural and economic importance (Orkwor, 1998; Bourke & Vlassak, 2004; Sukal et
al., 2015). Although yam production is highest in the African region, predominated by
Dioscorea rotundata-cayenensis and accounting for 95% of the world total production
(FAOSTAT, 2018), yam is also very important in the Pacific, where D. alata and D.
esculenta are the dominant species (Kenyon et al., 2008). Yam cultivation is almost
exclusively through vegetative propagation, which facilitates the vertical transmission
of viruses, leading to virus accumulation and associated production losses. Virus
accumulation presents major challenges in the exchange of yam germplasm (Kenyon
et al., 2008; Seal et al., 2014; Sukal et al., 2015).
Badnaviruses, classified as a number of Dioscorea bacilliform virus (DBV)
species, are reported as being the most widespread of all the virus groups known to
infect yams (Eni et al., 2008a,b; Kenyon et al., 2008; Bousalem et al., 2009). DBVs
are vegetatively transmitted as well as vectored by several species of mealybugs
(family Pseudococcidae) in a semi-persistent manner (Phillips et al., 1999; Kenyon et
al., 2001; Atiri et al., 2003; Odu et al., 2004; Bömer et al., 2016). Although infection
of yams with DBVs has been associated with leaf symptoms such as veinal chlorosis,
necrosis, puckering and crinkling, symptomless infections can also occur (Phillips et
al., 1999; Lebot, 2009; Seal et al., 2014; Bömer et al., 2016).
At present, five species of yam-infecting badnaviruses, Dioscorea bacilliform
AL virus (DBALV), Dioscorea bacilliform RT virus 1 (DBRTV1), Dioscorea
bacilliform RT virus 2 (DBRTV2), Dioscorea bacilliform TR virus (DBTRV) and
Dioscorea bacilliform SN virus (DBSNV), are taxonomically accepted by the ICTV
141
(Adams & Carstens, 2012; Adams et al., 2018) and a further three species, Dioscorea
bacilliform AL virus 2 (DBALV2), Dioscorea bacilliform ES virus (DBESV) and
Dioscorea bacilliform RT virus 3 (DBRTV3) have been described (Sukal et al., 2017;
Bömer et al., 2018). However, additional studies using PCR suggest that there may be
at least another eight putative badnavirus species infecting yams worldwide (Kenyon
et al., 2008; Bousalem et al., 2009, Bömer et al., 2016).
The Centre for Pacific Crops and Trees (CePaCT) within the Pacific
Community (SPC) has a unique collection of yam germplasm from the Pacific region.
The potential use of this germplasm to address production issues, such as yield losses
from pests and diseases, as well as agronomic traits pertinent to export requirements,
is immense but remains unexplored due to the unavailability of suitable diagnostic
protocols for the detection of badnaviruses. Sukal et al., (2017) recently showed the
existence of three different DBVs (DBALV2, DBESV and DBRTV2) in Pacific
germplasm collections using RCA and sequencing. They further optimized the RCA
protocol to improve badnavirus amplification and showed that RCA coupled with next
generation sequencing can be used as an effective tool for DBV amplification and
characterization (Sukal et al., 2018, manuscript in preparation). In this study, we
further apply this approach to characterize the molecular diversity of badnaviruses
infecting the Pacific yam germplasm conserved at SPC-CePaCT.
Methods
Sample details, total nucleic acid (TNA) extractions
SPC-CePaCT presently maintains a collection of 283 yam accessions in tissue
cultures. For this study, 224 of these accessions were established in SPC-CePaCT’s
insect-proof screenhouse. The remaining 59 yam accessions were not available for this
142
study, because they were in low numbers. After at least three months following
acclimatization, leaf samples were collected and total nucleic acids (TNA) extracted
using a CTAB extraction protocol described previously (Kleinow et al., 2009). The
purified TNA was quantified using a NanoDrop2000 spectrophotometer
(ThermoFisher Scientific, Australia) and the concentration adjusted to ~500 ng/μl with
sterile nuclease-free water (NF-H2O). DA/NGA01 an accession from Nigeria, virus
screened by IITA before being sent to SPC-CePaCT and experimentally determined
to be negative for episomal DBV was used as a negative control for the RCA screening.
Viral DNA enrichment, RCA-RFLP, cloning and Sanger sequencing
A badnavirus-biased RCA approach, described by Sukal et al., (2018;
manuscript in preparation), was used to enrich for viral circular DNA. Briefly, a
mixture of 32 degenerate badnavirus primers at a final concentration of 0.4 μM of each
primer, 1 × phi29 buffer (NEB, Australia) and 1 μl (~500 ng) of TNA was made up to
a final volume of 10 μl with sterile NF-H2O and denatured at 95°C for 3 min, cooled
to 4°C and placed on ice. Ten μl of reaction mixture consisting of 2.5 μM exo-resistant
random hexamers (ThermoFisher Scientific), 1 × phi29 buffer, 2 ng/μl bovine serum
albumin (BSA), 4 mM DTT, 15 mM dNTPs, 5 U/μl of phi29 DNA polymerase
(ThermoFisher Scientific) and sterile NF-H2O to make up the final volume, was
prepared and added to each denatured sample. Reactions were incubated at 36°C for
18 h, followed by 65°C for 10 min to denature the phi29 polymerase.
RCA products were digested using several restriction endonucleases
independently, including EcoRI and SphI (NEB, USA). The enzymes were selected
following in silico restriction analysis of published badnavirus genome sequences and
from experimental experience. Digest products were electrophoresed through 1.5%
143
agarose gels stained with SYBR® Safe (ThermoFisher Scientific) and fragments of
interest were excised, purified and cloned as described in Sukal et al. (2017). The
plasmids were screened using restriction analysis and insert-containing plasmids were
sequenced with universal M13F/R primers. The resulting reads were queried against
GenBank using the BLASTn and BLASTx algorithms and where sequence reads
matched to badnavirus genomes the cloned DNAs were further sequenced with
badnavirus degenerate primers BadnaFP/RP (Yang et al., 2003). A primer-walking
approach was subsequently used to sequence the complete genomes of four DBALV
isolates infecting three samples (one each of D. alata, D. transversa and D. trifida)
from Vanuatu as well as one D. esculenta sample from Tonga. The putative restriction
sites were confirmed with PCR using sequence-specific primers flanking the sites as
described previously (Sukal et al., 2017).
Next generation sequencing and genome assembly
RCA was carried out as described previously and undigested RCA products of 10 PNG
samples (all from D. alata) and six Vanuatu samples (including one from D. alata,
two from D. esculenta and three from D. bulbifera) were purified using the Illustra™
GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare, United Kingdom)
and sent to the Central Analytical Research Facility (CARF), Queensland University
of Technology, Brisbane, Australia for library preparation and sequencing using the
Illumina MiSeq system to generate paired end reads of 301 bp. A further 98 samples
that did not produce any apparent restriction profile following RCA restriction digest
with EcoRI were purified and pooled by country and sent for the preparation of 13
additional libraries, including six libraries for Fiji, two libraries each for Vanuatu, New
144
Caledonia and Federated States of Micronesia (FSM), and one library for PNG, with
subsequent sequencing by NGS as described previously.
Quality of the raw reads was assessed with FastQC v0.10.1 (Babraham
Bioinformatics, UK). A pipeline similar to that described in Muller et al. (2018) for
the processing of NGS data and characterization of badnavirus diversity from cacao
was used in this study. Raw reads were trimmed to obtain optimum quality using the
dynamic trim function of SolexaQA++ v.3.1.3 (Cox et al., 2010) and FASTX-Toolkit
(http://hannonlab.cshl.edu/fastx_toolkit/). Quality corrected reads were then mapped
against the D. rotundata reference genome (GenBank accessions DF933857-
DF938579; Tamiru et al., 2017) using the Geneious® v11.0.2
(http://www.geneious.com; Kearse et al., 2012) reference mapper algorithm with
default settings. Unmapped reads were assembled into contigs using the SPAdes
v3.5.0 algorithm (Bankevich et al., 2012) with k-mers 21, 33 and 55 on the Galaxy
platform (Afgan et al., 2015). The resulting contigs were imported into Geneious and
BLASTn analysis was performed against a local database of all known caulimoviridae
complete genome sequences available on NCBI as of April 2018. Finally, the viral
genome having the highest homology with each assembled contig was then used to
perform reference guided mapping to generate a consensus viral genome. Geneious
was used to manually examine each assembled genome and ORF prediction on the
plus-strand of the putative viral genomes was carried out using Geneious and
ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/) to determine putative viral open
reading frames (ORFs) and identify conserved badnaviral sequence motifs.
145
Pairwise sequence comparisons and phylogenetic analysis
Partial reverse transcriptase (RT)-ribonuclease H (RNase H) coding sequences
delimited by the BadnaFP/RP (Yang et al., 2003) primers, representing the region
accepted for species demarcation of badnaviruses by the ICTV (Geering and Hull,
2012), were used to determine the pairwise nucleotide sequence identity using the
Sequence Demarcation Tool (SDTv1.2) (Muhire et al., 2014) for the full-length
genomes generated. For phylogenetic analysis, multiple sequence alignments were
constructed using ClustalW (Larkin et al., 2007) within MEGA7 (Kumar et al., 2016)
and the Maximum-Likelihood method (Kimura-2-parameter model) used to
reconstruct phylogenetic trees following 1000 bootstrap iterations.
Results
RCA and Sanger sequencing
TNA was extracted from 224 yam samples representing five yam species, including
D. alata (185), D. esculenta (31), D bulbifera (6), and one each of D. transversa and
D. trifida. All extracts were screened for putative badnavirus infection using RCA
followed by independent restriction digestion with EcoRI and SphI. In total, 35
samples from three countries, representing five yam species, produced restriction
profiles indicative of badnaviral genome amplification, while none of the 42, 21 and
24 D. alata samples from FSM, Fiji or New Caledonia, respectively, and 17, two and
one D. esculenta samples from Fiji, PNG or Samoa, respectively, produced profiles
indicative of badnavirus infection (Table 1).
146
Table 1 Summary of badnavirus RCA testing results
D. alata D. bulbifera D. esculenta D. transversa D. trifida
No. of
acc.
No.
tested
No.
positive
No. of
acc.
No.
tested
No.
positive
No. of
acc.
No.
tested
No.
positive
No. of
acc.
No.
tested
No.
positive
No. of
acc.
No.
tested
No.
positive
Fiji 43 42 0 0 0 0 20 17 0 0 0 0 0 0 0
FSM 24 21 0 0 0 0 0 0 0 0 0 0 0 0 0
PNG 44 38 15 0 0 0 6 2 0 0 0 0 0 0 0
New Caledonia 40 24 0 0 0 0 0 0 0 0 0 0 0 0 0
Samoa 4 2 0 1 1 0 1 1 0 0 0 0 0 0 0
Tonga 1 0 0 0 0 0 3 2 2 0 0 0 0 0 0
Vanuatu 73 58 2 7 5 5 11 9 9 1 1 1 1 1 1
Total 229 185 17 8 6 5 41 31 11 1 1 1 1 1 1
No. of acc. = Number of accessions, No. tested = number of accessions tested for badnavirus and No. of positive = number of accessions
badnavirus positive.
147
Of the 185 D. alata accessions tested, 15/38 samples from PNG and 2/58
samples from Vanuatu produced digest profiles indicative of badnavirus infection
following digestion of RCA-amplified DNA. In these 17 accessions, digestion using
EcoRI produced between three and seven fragments of less than 4 kb (represented in
Fig. 1A and 2A), while digestion using SphI produced a single 8 kb band in 15
accessions (represented in Fig. 1B and 2B). In accessions DA/PNG58, SphI digestion
produced two bands of 1 and 6 kb (Fig. 1B, lane 8), while in accession DA/VUT38,
SphI digestion produced three bands of 2.5, 3.5 and 5 kb (Fig. 2B, lane 2).
Five distinct EcoRI restriction profiles (hereafter referred to as P1-P5) were
observed in digests of RCA-amplified DNA from the D. alata samples from PNG (Fig.
1A). In eight samples (DA/PNG06, 20, 22, 23, 43, 51, 57and 59), digestion using
EcoRI produced profile P1 with seven fragments of 0.3, 0.5, 0.6, 0.8, 1.0, 1.6 and 3
kb (Fig. 1A, lane 1-2), while in two samples (DA/PNG07 and 45) digestion using
EcoRI produced profile P2 with seven fragments of 0.6. 0.8, 1.1, 1.6, 2.5, 3 and 4 kb
(Fig. 1A, lane 3-4). Three samples (DA/PNG05, 12 and 14) showed profile P3 with
six fragments of 0.5, 0.8, 1.0, 1.6, 2.3 and 3 kb (Fig. 1A, lanes 5-6), sample
DA/PNG17 showed profile P4 with three fragments of 1.4, 2.5, and 4 kb (Fig. 1A
lane 7) and sample DA/PNG58 produced profile P5 with four fragments of 0.6, 0.8,
2.5 and 4 kb (Fig. 1A, lane 8). Digestion of the 15 RCA positive PNG D. alata samples
using SphI produced a single 8 kb band in 14 of the accessions, while SphI digestion
of RCA-amplified DNA from accession DA/PNG58 produced two bands of 1 and 6
kb (Fig. 1A, lane 8).
148
Figure 1 (A) EcoRI and (B) SphI restriction analysis of PNG D. alata RCA positive
samples. Lane 1-8 are DA/PNG20, DA/PNG22, DA/PNG07, DA/PNG45,
DA/PNG12, DA/PNG14, DA/PNG17 and DA/PNG58. Lane 9 is a known negative
sample (DA/NGA01) and Lane 10 is a no template control. M – GeneRuler 1 kb DNA
ladder (ThermoFisher Scientific).
(A)
(B)
149
Figure 2 (A) EcoRI and (B) SphI restriction analysis of Vanuatu (VUT) and Tonga
(TON) RCA positive samples. Lanes 1-12 are samples DA/VUT12, DA/VUT38,
DB/VUT01, DB/VUT02, DB/VUT04, DB/VUT07, DE/VUT01, DE/VUT02,
DE/TON02, DE/TON03, DTV/VUT01 and DTF/VUT01. Lane 13 is a known
negative sample (DA/NGA01) and Lane 14 is a no template control. M – GeneRuler
1 kb DNA ladder (ThermoFisher Scientific). DA - D. alata, DB - D. bulbifera, DE –
D. esculenta, DTV – D. transversa and DTF – D. trifida.
(A)
(B)
150
The two D. alata samples from Vanuatu which tested positive also produced
distinct restriction profiles following digestion using EcoRI, with sample DA/VUT12
producing four fragments of 1.0, 1.2, 1.7 and 3.3 kb (Fig. 2A, lane 1), while sample
DA/VUT38 produced five fragments of 1.0, 1.2, 1.7, 2.7 and 3.5 kb (Fig. 2A, lane
2). Digestion of RCA-amplified DNA from accession DA/VUT12 using SphI
produced a single 8 kb band (Fig. 2B, lane 1), while in accession DA/VUT38 SphI
digestion produced 3 bands of 2.5, 3.5 and 5 kb (Fig. 2B, lane 2).
Of the six D. bulbifera accessions tested, all five from Vanuatu produced
restriction profiles indicative of badnavirus infection, producing six to seven
fragments of less than 4 kb following digestion with EcoRI (Fig. 2A, lanes 3-6) or a
single putative full-length digest fragment using SphI (Fig. 2B, lanes 3-6), while the
single accession tested from Samoa produced no visible digest fragments. Two distinct
restriction profiles were observed following EcoRI digestion of the RCA-amplified
DNA from D. bulbifera accessions from Vanuatu. Three samples (DB/VUT01, 02 and
03) produced a profile with seven fragments of 0.9, 1.0, 1.1, 1.2, 1.7, 3.0 and 3.4 kb
(Fig. 2A, lane 3-4), while digestion of two samples (DB/VUT04 and 07) produced six
fragments of 0.9, 1.0, 1.2, 1.7, 3.4 and 4.0 kb (Fig. 2A, lanes 5-6).
Of the 31 D. esculenta accessions tested, 11 showed profiles indicative of
badnavirus infection including two samples from Tonga and all nine samples tested
from Vanuatu, while none of the 17, two and one accessions from Fiji, PNG and
Samoa, respectively, produced any visible digest fragments. EcoRI digestion of RCA-
amplified DNA from the nine positive D. esculenta samples from Vanuatu
(DE/VUT01-09) (represented in Fig. 2A, lanes 7-8) and two positive D. esculenta from
Tonga (DE/TON02-03) (Fig. 2A, lanes 9-10) resulted in the same restriction profile as
previously observed in accession DA/VUT12, (Fig. 2A, lane 1), while digestion with
151
SphI produced a single 8 kb fragment for all 11 positive D. esculenta samples
(represented in Fig. 2B, lanes 7-8 and Fig. 2B, lanes 9-10, respectively).
When RCA-amplified DNA from the single D. transversa accession
(DTV/VUT01) was digested using EcoRI (Fig. 2A, lane 11), the profile matched that
of the Vanuatu D. bulbifera samples DB/VUT04 and 07 (Fig. 2A, lane 5 and 6).
Similarly, the restriction profile of the EcoRI digested RCA-amplified DNA from the
single D. trifida accession (DTF/VUT01) matched that of the D. alata accession
DA/VUT12 as well as the D. esculenta accessions from Vanuatu and Tonga described
previously (Fig. 2A, lane 12).
The SphI-digested RCA products of the 35 previously uncharacterized samples
were all cloned and sequenced using M13F/R primers, and subsequently using
BadnaFP/RP primers. BLASTn analysis of the partial RT/RNase H sequences
obtained following sequencing with the BadnaFP/RP primers revealed that all 15
sequences originating from PNG showed 89-100% nucleotide identity with DBALV2
isolate PNG10 (GenBank accession KY827395), while the 18 sequences from
Vanuatu and two sequences from Tonga showed 92-100% nucleotide identity with
DBALV isolate 2ALa (GenBank accession KX008571).
The complete genome sequences of DBALV were subsequently obtained
using a primer-walking approach from D. alata accession DA/VUT12, D. transversa
accession DTV/VUT01 and D. trifida accession DTF/VUT01 from Vanuatu, as well
as D. esculenta sample DE/TON02 from Tonga. The complete genome sizes of these
four virus isolates comprised 7531, 7503, 7579 and 7390 bp, respectively, and the
complete annotated sequences were submitted to GenBank under accession numbers
MH404165- MH404168. In order to identify the sequences of all templates amplified
152
using RCA, the remainder of the samples were analysed using a RCA-NGS approach
and screened for circular DNA viruses.
Next generation sequencing of Pacific DBV isolates
Thirty five out of 224 accessions produced RCA restriction profiles indicative
of the presence of badnavirus. Sixteen of these 35 samples, including ten D. alata
samples from PNG and six samples from Vanuatu (one D. alata, two D. esculenta and
three D. bulbifera) were selected based on differences in EcoRI restriction profiles,
partial RT/RNase H-coding nucleotide sequences, host species or the country of origin,
and undigested RCA products were sequenced using NGS. In addition, undigested
RCA products from a further 98 samples that did not produce any visible restriction
profiles following digestion of RCA products with EcoRI and SphI were purified and
pooled by countries and/or host plant species, and also sent for library preparation and
NGS. These 98 samples were pooled into 13 libraries consisting of six libraries from
Fiji (four D. alata and two D. esculenta), two each from Vanuatu (D. alata), New
Caledonia (D alata) and FSM (D. alata), with each library consisting of eight pooled
samples, and one library consisting of two pooled D. esculenta samples from PNG.
Raw NGS paired-end reads were quality corrected, host genomic DNA subtracted, and
the reads de novo assembled.
Using the NGS data from the sixteen individual samples (10 from PNG and six
from Vanuatu), 16 complete badnavirus genomes were assembled. All of the complete
genomes generated from PNG samples (Table 2) were most similar to DBALV2, while
all of the complete genomes assembled from Vanuatu samples (Table 3) were most
similar to DBALV. The 16 complete genomes were annotated and submitted to
GenBank under the accessions MH404155-MH404164 and MH404169-MH404174.
153
The NGS data obtained from the 13 pooled libraries resulted in contigs with no
significant homology to viral sequences, consistent with the RCA restriction analysis.
Analysis of DBALV and DBALV2 complete genomes
A total of 20 complete genome sequences were obtained using either NGS or Sanger
sequencing of RCA products from 20 individual accessions. Sixteen of the genomes
were generated using NGS, of which 10 were DBALV2 originating from PNG and the
remaining 6 were DBALV originating from Vanuatu. The four genomes generated
using Sanger sequencing were all DBALV including three samples originating from
Vanuatu and one sample from Tonga. Consistent with the RCA restriction analysis
(Fig. 1 and 2), all of the complete genomes contained between three and seven EcoRI
sites and a single SphI site, except for samples DA/PNG58 and DA/VUT38 which had
two SphI sites.
Dioscorea bacilliform AL virus (DBALV) from the Pacific
Using NGS, six complete DBALV genomes from Vanuatu were generated
including one from D. alata, three from D. bulbifera and two from D. esculenta, while
four complete genomes were generated using Sanger sequencing including one each
from D. alata, D. transversa and D. trifida from Vanuatu as well as one complete
genome from D. esculenta originating from Tonga. The complete genomes ranged
from 7390 to 7579 bp in length with their GC contents varying between 42.8 and
43.7%. All contained three ORFs typical of badnaviruses with ORF 1 432 bp in length,
ORF 2 378 bp in length and ORF 3 ranging from 5676-5736 bp in length (Table 3).
154
Table 2 Genomic features of DBALV2 isolates obtained from sequencing of RCA products
Virus Isolate Genome ORF 1 ORF 2 ORF 3
Length (bp) Length Start-stop (frame) Length Start-stop (frame) Length Start-stop (frame)
(% G + C) (bp) (codon use) (bp) (codon use) (bp) (codon use)
DBALV2-PNG07_DA 7843 429 773-1201 (+2) 396 1198-1593 (+1) 5811 1590-7400 (+3) 42.1
(ATG-TGA)
(ATG-TGA)
(ATG-TGA)
DBALV2-PNG12_DA 7860 447 754-1200 (+1) 396 1197-1592 (+3) 5844 1592-7435 (+2) 42.8
(CTG-TGA)
(ATG-TAA)
(ATG-TAA)
DBALV2-PNG14_DA 7862 447 754-1200 (+1) 396 1197-1592 (+3) 5847 1592-7438 (+2) 42.7
(CTG-TGA)
(ATG-TAA)
(ATG-TAA)
DBALV2-PNG20_DA 7879 429 774-1202 (+3) 399 1199-1597 (+2) 5856 1594-7449 (+1) 41.0
(ATG-TGA)
(ATG-TGA)
(ATG-TGA)
DBALV2-PNG22_DA 7877 429 775-1203 (+1) 399 1200-1598 (+3) 5852 1595-7447 (+2) 41.0
(ATG-TGA)
(ATG-TGA)
(ATG-TAA)
DBALV2-PNG23_DA 7863 447 754-1200 (+1) 396 1197-1592 (+3) 5847 1592-7438 (+2) 42.7
(CTG-TGA)
(ATG-TAA)
(ATG-TAA)
DBALV2-PNG45_DA 7849 441 760-1200 (+1) 396 1197-1592 (+3) 5838 1592-7429 (+2) 42.7
(CTG-TGA)
(ATG-TAA)
ATG-TAA
DBALV2-PNG51_DA 7876 429 774-1202 (+3) 399 1199-1597 (+2) 5853 1594-7446 (+1) 41.0
(ATG-TGA)
(ATG-TGA)
(ATG-TAA)
DBALV2-PNG58_DA 7876 429 774-1202 (+3) 399 119-1597 (+2) 5853 1594-7446 (+1) 41.0
(ATG-TGA)
(ATG-TGA)
ATG-TAA
DBALV2-PNG59_DA 7860 447 754-1200 (+1) 396 1197-1592 (+3) 5844 1592-7435 (+2) 42.8
(CTG-TGA)
(ATG-TAA)
(ATG-TAA)
155
Table 3 Genomic features of DBALV isolates obtained from sequencing of RCA products
Virus Isolate Genome ORF 1 ORF 2 ORF 3
Length (bp) Length Start-stop (frame) Length Start-stop (frame) Length Start-stop (frame)
(% G + C) (bp) (codon use) (bp) (codon use) (bp) (codon use)
DBALV-TON02_DE* 7390 432 447-878 (+3) 378 875-1252 (+2) 5700 1252-6951 (+1) 43.1
(ATG-TGA)
(ATG-TAA)
(ATG-TAA)
DBALV-VUT12_DA* 7531 432 589-1020 (+1) 378 1017-1394 (+3) 5700 1394-7093 (+2)
43.7 (ATG-TGA) (ATG-TAA) (ATG-TAA)
DBALV-VUT38_DA 7523 432 576-1007 (+2) 378 1004-1381(+1) 5670 1381-7050 (+3) 43.1
(ATG-TGA)
(ATG-TAA)
(ATG-TAA)
DBALV-VUT01_DB 7512 432 571-1002 (+2) 378 999-1376 (+1) 5688 1376-7063(+3)
42.8 (ATG-TGA) (ATG-TAA) (ATG-TGA)
DBALV-VUT02_DB 7515 432 574-1005 (+1) 378 1009-1379 (+3) 5688 1379-7066 (+2) 42.8
(ATG-TGA)
(ATG-TAA)
(ATG-TGA)
DBALV-VUT04_DB 7502 432 576-1007 (+2) 378 1004-1381 (+1) 5676 1381-7056 (+3) 43.0
(ATG-TGA)
(ATG-TAA)
(ATG-TGA)
DBALV-VUT01_DE 7520 432 577-1008 (+1) 378 1005-1382 (+3) 5700 1382-7081 (+2)
43.0 (ATG-TGA) (ATG-TAA)
(ATG-TAA)
DBALV-VUT04_DE 7401 432 476-907 (+2) 378 904-1281 (+1) 5682 1281-6962 (+3)
43.1 (ATG-TGA) (ATG-TAA) (ATG-TAA)
DBALV-VUT01_DTF* 7579 432 600-1031 (+3) 378 1028-1405 (+2) 5736 1405-7140 (+1)
43.0 (ATG-TGA) (ATG-TAA) (ATG-TAA)
DBALV-VUT01_DTV* 7502 432 583-1014 (+1) 378 1011-1388 (+3) 5676 1388-7063 (+2)
42.8 (ATG-TGA) (ATG-TAA) (ATG-TGA)
*Sequence was obtained with Sanger sequencing.
156
The single intergenic region (IR), varying from 885-1038 bp in length, contained
several conserved motifs typical of plant dsDNA viruses (Benfey & Chua, 1990;
Medberry & Olszewski, 1993). The putative tRNAmet binding site (5`-
TGGTATCAGAGCTCGGTT-3`) with 88.9% nucleotide identity to the plant tRNAmet
consensus sequence (3`-ACCAUAGUCUCGGUCCAA-5`), which has been
described as the priming site for reverse transcription, was designated as the origin of
the circular genomes consistent with the convention used for other badnaviruses. In
addition, a TATA-box (TATATAA) and polyadenylation signal, analogous to the 35S
promotor of cauliflower mosaic virus (CaMV), were also identified in the region 5` of
the tRNAmet site in all genomes.
BLASTn analysis of the full-length genome sequences confirmed that all had
highest nucleotide identity to DBALV (GenBank accessions KX008571, KX008572
and KX008573) with nucleotide sequence identity ranging from 89.2 to 90%. BLASTn
analysis using the partial RT/RNase H-coding region of each sequence revealed 97.5
to 99.8% nucleotide identity to partial RT/RNase H-coding sequences generated
previously from Vanuatu yams (GenBank accessions AM072705 to AM072708). The
partial RT/RNase H-coding sequence delineated by the BadnaFP/RP primers was
identified in silico and used to carry out pairwise sequence analysis (PASC) and
phylogenetic comparison amongst the sequences and with other published DBALV
sequences. PASC revealed that DBALV sequences from Vanuatu and Tonga had 90.5
to 100% nucleotide identity amongst each other and 85 to 94% to previously published
isolates from Africa (Fig. 3A). Phylogenetic analysis using the partial RT/RNase H-
coding region revealed that the DBALV isolates from Africa were ancestral to the
isolates from the Pacific (Fig. 3B).
157
Figure 3 PASC and phylogenetic analysis using partial RT/RNase H-coding nucleotide sequences showing the relationships of DBALV
isolates from this study with previously published complete DBALV sequences. (A) Pairwise nucleotide sequence identities were
determined using the Sequence Demarcation Tool (SDTv1.2) (Muhire et al., 2014). (B) Maximum-Likelihood phylogenetic tree
constructed with the Kimura-2-parameter model, using DBALV2-PNG10 (KY827395) as the outgroup. VUT-Vanuatu, TON-Tonga,
PNG-Papua New Guinea, while KX008571, KX008572 and KX008595 are from Africa.
158
Dioscorea bacilliform AL virus 2 (DBALV2)
RCA combined with NGS was used to generate 10 complete sequences of PNG
badnavirus sequences derived from D. alata. The complete genomes ranged from 7860
to 7879 bp in length and had a GC content of 41 to 42.8%. Consistent with DBALV2
isolate PNG10, the sequences contained three ORFs with ORF 1 varying in length
from 429-447 bp, ORF 2 varying in length from 396-399 bp and ORF 3 varying in
length from 5811-5856 bp (Table 2). Conserved motifs including the tRNAmet site (5`-
TGGTATCAGAGCKYGGTT-3`, underlined nucleotides were not conserved in all
sequences), TATA-boxes (TATATAA) and polyadenylation signals were identified in
the IR, similar to those present in the DBALV sequences.
BLASTn analysis of the full genome sequences revealed a sequence identity
of 87 to 89.1% to the previously published DBALV2 isolate PNG10 (GenBank
accession KY827395) originating from PNG, while BLASTn analysis using the partial
RT/RNase H-coding region nucleotide sequences revealed 90.7 to 97.5% nucleotide
identity to partial RT/RNase H sequences generated previously from PNG D. alata
(GenBank accessions AM072674, AM072683 and AM072685). PASC and
phylogenetic analysis of DBALV2 was carried out using the partial RT/RNase H-
coding sequences as described earlier. PASC between the DBALV2 sequences
identified in this study, together with the previously published complete DBALV2-
PNG10 sequence from PNG, revealed 88.8-100% nucleotide identity between the
sequences (Fig. 4A), with the DBALV2-PNG10 sequence appearing to be ancestral to
the 10 sequences characterized in this study (Fig. 4B).
159
Figure 4 PASC and phylogenetic analysis using partial RT/RNase H-coding nucleotide sequences showing the relationships of DBALV2
isolates from this study with previously published complete DBALV2 sequences. (A) Pairwise nucleotide sequence identities were
determined using the Sequence Demarcation Tool (SDTv1.2) (Muhire et al., 2014). (B) Maximum-Likelihood phylogenetic tree
constructed with the Kimura-2-parameter model, using DBALV-[2ALa] (KX008571) as the outgroup. PNG-Papua New Guinea, while
KX008571 is from Africa.
160
Discussion
In this study, we have characterized the sequence diversity of DBVs present in
the Pacific yam germplasm collections held in trust by SPC-CePaCT. A subset of 224
accessions from the collection was screened with RCA, with subsequent restriction
analysis indicating the presence of episomal badnavirus in 35 samples. Based on
differences in EcoRI restriction profiles, partial RT/RNase H-coding nucleotide
sequences, host species or the country of origin, a total of 20 complete DBALV and
DBALV2 genome sequences were obtained from five different yam species
originating from PNG, Tonga and Vanuatu.
Based on this, and other studies (Kenyon et al., 2008), DBALV appears to be
the most prevalent badnavirus in Pacific yam germplasm with the virus identified in
five yam species (D. alata, D. bulbifera, D. esculenta, D. transversa and D. trifida)
from Vanuatu as well as D. esculenta from Tonga. Using a PCR-based approach,
Kenyon et al. (2008) reported the presence of DBALV from Vanuatu and Solomon
Islands. Due the unavailability of samples, the occurrence of DBALV in Solomon
Islands could not be confirmed in the present study. Whereas a very low prevalence
(3.4%) of DBALV was found in D. alata accessions from Vanuatu, all D. esculenta
and D. bulbifera samples tested were found to be infected with the virus. The low
prevalence of DBALV in Vanuatu D. alata could be due to the fact that part of the
collection tested herein (31/73) was sent to SPC-CePaCT ex Centre de Coopération
Internationale en Recherche Agronomique pour le Développement (CIRAD)
following virus screening, including testing for DBV, and only the virus negative
accessions were sent to SPC-CePaCT to be included in the yam collection (Filloux et
al., 2006). In the current study, the accessions from CIRAD were also found to be
DBV negative. Further, the previously untested Vanuatu D. alata accessions were also
161
found to have a low prevalence of DBALV (2/42), which correlates with the results of
Filloux et al (2006) who also reported a low incidence of DBALV in Vanuatu D. alata
with only 4/56 accessions testing positive in the previous study. Additionally, none of
the samples from Fiji, FSM, PNG, New Caledonia or Samoa tested in the present study
were positive for DBALV.
DBALV2 was only detected from PNG D. alata samples in the present study,
with an incidence of 42.5%, which was significantly higher than the incidence of
DBALV infection of D. alata from Vanuatu. Kenyon et al. (2008) also reported
DBALV2 from D. alata accessions only, however, they detected DBALV2 in samples
from both PNG and Vanuatu. In contrast, DBALV2 was not detected from the Vanuatu
samples or from other countries included in this study.
DBESV has been previously reported and characterized from a D. esculenta
accession from Fiji (DE/FJ14) (Sukal et al., 2017). Testing of additional yam samples
in the present study failed to detect DBESV from any additional D. esculenta
accessions, or accessions of other yam species from either Fiji or any other countries
included in the study. Previously, Kenyon et al. (2008) reported DBESV-like
RT/RNase H partial sequences from D. alata accessions originating from Fiji, PNG,
Tonga and Vanuatu, as well as D. esculenta accessions originating from Fiji, PNG and
Solomon Islands. However, no additional accessions (other than DE/FJ14 described
previously) tested positive for DBESV, therefore, as speculated by Kenyon et al.,
(2008), DBESV may be present as an endogenous sequence in some yam species.
Since the focus of the present study was to characterize the episomal badnavirus
sequences present in yam, the presence of endogenous DBESV was not determined
but is worthy of future investigation.
162
DBRTV2 has been previously characterized from a single Samoan D.
rotundata accession (Sukal et al., 2017). Bömer et al. (2016) also detected and
characterized DBRTV2 from D. rotundata originating from Nigeria. Therefore, it is
possible that DBRTV2 is restricted to D. rotundata. However, since the SPC-CePaCT
collections do not include D. rotundata from any additional Pacific countries, the
presence of DBRTV2 in countries other than Samoa could not be ascertained. Further
field collections and testing should be done to determine whether DBRTV2 is more
widely prevalent in the Pacific and whether it occurs in other yam species. Since D.
rotundata originates from West Africa (Lebot, 2009), it could be postulated that
DBRTV2 may also originate from West Africa and was distributed into the Pacific
with the historical introduction of D. rotundata accessions.
RCA has been successfully used to characterize badnaviruses infecting banana
(James et al., 2011; Wambulwa et al., 2012; Wambulwa et al., 2013; Baranwal et al.,
2014; Carnelossi et al., 2014; Javer-Higginson et al., 2014; Sharma et al., 2014, 2015),
cacao (Chingandu et al., 2017a,b; Muller et al., 2018), fig (Laney et al., 2012),
mulberry (Chiumenti et al., 2016), Rubus spp. (Diaz-Lara et al., 2015) and yam
(Umber et al., 2014; Bömer et al., 2016, 2018; Sukal et al., 2017). RCA in combination
with NGS has also been shown to be very effective in characterizing badnaviruses
infecting cacao (Chingandu et al., 2017a,b; Muller et al., 2018). The present study has
further demonstrated the utility of RCA-NGS for whole genome amplification and
characterization of badnaviruses from yam. Although RCA-NGS can be effectively
used for virus detection and characterization, the high costs currently associated with
NGS makes routine diagnosis by this method impractical. However, with improved
specificity and the increasing number of badnavirus sequences available from yam,
RCA is now an important consideration for badnavirus detection.
163
During restriction analysis of RCA using EcoRI and SphI, the restriction
profiles generated using EcoRI were found to vary considerably between virus isolates
in the different yam species studied (Fig. 1A and 2A). Therefore, it may be necessary
to use a combination of enzymes with single or multiple recognition sites within the
virus genome for diagnostic purposes. The use of an enzyme with multiple recognition
sites would increase the chances of detection, since the loss of a site through genetic
changes during virus replication would be unlikely to affect multiple sites for a single
enzyme. In contrast, the use of an enzyme with a single recognition site may be
significantly affected by errors resulting during genome replication, since the loss of a
single site would result in no detection following restriction analysis of RCA products.
We also found that other enzymes, such KpnI and StuI, can also complement EcoRI
and SphI in determining a DBV positive sample (data not shown).
Restriction sites both within and between DBV species were found to be
variable. For example, EcoRI restriction analysis of PNG D. alata RCA products
resulted in five distinct restriction profiles (Fig. 1A, lanes 1-8). Similarly, EcoRI
restriction analysis of Vanuatu D. alata, D. bulbifera, D. esculenta, D. transversa and
D. trifida and Tonga D. esculenta resulted in an additional four distinct profiles.
However, sequencing of the RCA products from PNG samples having the different
restriction profiles revealed that they were DBALV2, while all of the products from
the Tonga and Vanuatu were DBALV. In some of these profiles, the EcoRI restriction
fragments total greater than 8 kb, for example DA/PNG07 and 45 (Fig. 1A, lanes 3-
4), DA/VUT38 (Fig. 2A, lane 2), DB/VUT01, 02 and 03 (Fig. 2A, lanes 3-4),
DB/VUT04 and 07 (Fig. 2A, lanes 5-6), (DTV/VUT01) (Fig. 2A, lane 11),
DTF/VUT01 (Fig. 2A, lane 12), which may be indicative of the presence of multiple
viral sequences. However, sequencing revealed that these accessions contained only
164
single DBV infections. Possible explanations for this observation include star activity
of EcoRI, the existence of virus sequence variants within an accession, or co-
amplification of plant genomic DNA during RCA or partial digestion of RCA-
amplified DNA. Star activity of EcoRI and/or partial digests is unlikely due to both
the use of high-fidelity enzyme and the observation that the restriction profiles were
reproducible when RCA-amplified DNA from samples was digested on different
occasions. Furthermore, some of these profiles were consistent between multiple
samples infected with either of the two virus species. The amplification of detectable
levels of plant genomic DNA is also unlikely as we used a directed RCA approach,
which has been shown to have improved specificity towards badnaviral genome
amplification in preference to host genomic DNAs (Sukal et al., 2018 manuscript in
preparation). Sequencing of visible digest fragments confirmed that they were all of
viral origin and not derived from host plant genomic DNA. Therefore, it is likely that,
in some samples, DBVs are probably present as a collection of sequence variants or
quasispecies which produce slightly variable digest patterns using some enzymes, such
as EcoRI.
These results suggest that while an enzyme with multiple sites, such as EcoRI,
may not conclusively identify the virus species present in all cases, when combined
with single or double cutting enzymes such as SphI it can be effectively used to
confirm the presence of badnavirus through the observation of profiles which clearly
indicate the presence of a single 8 kb fragment, or two fragments which total 8 kb. In
this way, RCA will be an effective tool for determining the presence/absence of
episomal DBVs in yams, which is sufficient for use in germplasm indexing. Further,
the expanding knowledge of episomal integrated sequence diversity of badnavirus will
165
support the development of targeted diagnostic procedures such as PCR using virus
specific-primers.
This study, together with our previous work on yam badnavirus
characterization (Sukal et al., 2017; Sukal et al., 2018 manuscript in preparation)
significantly increases the knowledge on yam infecting badnaviruses in the Pacific,
with 24 complete genome sequences belonging to four different species, infecting six
species of yam from five countries, now available. Prior to this study, an additional
Caulimoviridae member that is distantly related to badnaviruses, from Samoan D.
nummularia accessions (DN/SAM01 and 02), was also identified and characterized
(Sukal et al., 2018). Collectively, these studies on Pacific yams show that the dominant
yam badnaviruses present in Pacific germplasm differs from that of the African region.
Although some virus species, such as DBALV, are present in both regions, many of
the viruses identified in the African region are not present in the Pacific and vice versa.
The present study shows that some of these viruses are restricted to only one or a few
countries in the Pacific and that special considerations must be taken to ensure that
germplasm collections are thoroughly screened to prevent the dissemination of these
badnavirus species to other countries. The availability of virus-tested yam germplasm
is essential for the effective distribution and eventual utilization of yams for improved
food and nutritional security in the Pacific.
166
Acknowledgements
The authors would like to thank the Centre for Pacific Crops and Trees (CePaCT) of
the Pacific Community (SPC) for making their yam collections available for this
project. Authors would also like to thank Dr. Michael Furlong and Dr. Grahame
Jackson for their support and advice on this research. The funding for the project was
provided by the Australian Centre for International Agricultural Research
(#PC/2010/065). AS is a John Allwright Fellowship recipient.
167
References
Adams MJ, Carstens EB, 2012. Ratification vote on taxonomic proposals to the
International Committee on Taxonomy of Viruses (2012). Archives of Virology
157, 1411–22.
Adams MJ, Lefkowitz EJ, King AMQ et al., 2018. Changes to taxonomy and the
international code of virus classification and nomenclature ratified by the
International Committee on Taxonomy of Viruses (2017). Archives of Virology
162, 2505–38.
Afgan E, Sloggett C, Goonasekera N et al., 2015. Genomics virtual laboratory: A
practical bioinformatics workbench for the cloud. PLoS ONE 10, e0140829.
Atiri GI, Winter S, Alabi OJ, 2003. Yam. In: Loebenstein G, Thottappilly G, eds. In:
Loebenstein G, Thottappilly G, eds. Virus and Virus-like Diseases of Major
Crops in Developing Countries. Dordrecht, Netherlands: Kluwer, 249–50.
Bankevich A, Nurk S, Antipov D et al., 2012. SPAdes: A new genome assembly
algorithm and its applications to single-cell sequencing. Journal of
Computational Biology 19, 455–77.
Baranwal VK, Sharma SK, Khurana D, Verma R, 2014. Sequence analysis of shorter
than genome length episomal banana streak OL virus like sequences isolated from
banana in India. Virus Genes 48, 120–7.
Benfey PN, Chua N-H, 1990. The Cauliflower mosaic virus 35s promoter:
combinatorial regulation of transcription in plants. Science 250, 959–66.
Bömer M, Rathnayake AI, Visendi P, Silva G, Seal SE, 2018. Complete genome
sequence of a new member of the genus Badnavirus, Dioscorea bacilliform RT
virus 3, reveals the first evidence of recombination in yam badnaviruses. Archives
of Virology 163, 533–8.
168
Bömer M, Turaki A, Silva G, Kumar P, Seal S, 2016. A Sequence-independent
strategy for amplification and characterization of episomal badnavirus sequences
reveals three previously uncharacterized yam badnaviruses. Viruses 8, 188.
Bourke RM, Vlassak V, 2004. Estimates of food crop production in Papua New
Guinea. Australian National University: Canberra.
Bousalem M, Durand O, Scarcelli N et al., 2009. Dilemmas caused by endogenous
pararetroviruses regarding the taxonomy and diagnosis of yam (Dioscorea spp.)
badnaviruses: Analyses to support safe germplasm movement. Archives of
Virology 154, 297–314.
Carnelossi PR, Bijora T, Facco CU et al., 2014. Episomal detection of banana streak
OL virus in single and mixed infection with cucumber mosaic virus in banana
‘Nanicão Jangada’. Tropical Plant Pathology 39, 342–6.
Chingandu N, Kouakou K, Aka R et al., 2017a. The proposed new species, cacao red
vein virus, and three previously recognized badnavirus species are associated
with cacao swollen shoot disease. Virology Journal 14, 199.
Chingandu N, Zia-ur-rehman M, Sreenivasan TN et al., 2017b. Molecular
characterization of previously elusive badnaviruses associated with symptomatic
cacao in the New World. Archives of Virology 162, 1363–71.
Chiumenti M, Morelli M, De Stradis A, Elbeaino T, Stavolone L, Minafra A, 2016.
Unusual genomic features of a badnavirus infecting mulberry. Journal of General
Virology 97, 3073–87.
Cox MP, Peterson DA, Biggs PJ, 2010. SolexaQA: At-a-glance quality assessment of
Illumina second-generation sequencing data. BMC Bioinformatics 11, 485.
Diaz-Lara A, Mosier NJ, Keller KE, Martin RR, 2015. A variant of rubus yellow net
virus with altered genomic organization. Virus Genes 50, 104–10.
169
Eni AO, Hughes JDA, Asiedu R, Rey MEC, 2008a. Sequence diversity among
badnavirus isolates infecting yam (Dioscorea spp.) in Ghana, Togo, Benin and
Nigeria. Archives of Virology 153, 2263–72.
Eni AO, Hughes JDA, Rey MEC, 2008b. Survey of the incidence and distribution of
five viruses infecting yams in the major yam-producing zones in Benin. Annals
of Applied Biology 153, 223–32.
FAOSTAT, 2018. Production Statistics (FAOSTAT). Rome, Italy: Food and
Agriculture Organization of the United Nations.
Filloux D, Girard J, Bgpi UMR, Ta K, Baillarguet CI De, 2006. Indexing and
elimination of viruses infecting yams (Dioscorea spp.) for the safe movement of
germplasm. In: Proc. 14th Triennial Symp. Trivandrum, India: ISTRC.
Geering ADW, Hull R. 2012. Family Caulimoviridae. In In: King AMQ, Adams MJ,
Carstens EB, Lefkowitz EJ, eds. Virus Taxonomy: Classification and
Nomenclature of Viruses - Ninth Report of the International Committee on
Taxonomy of Viruses. Oxford, UK: Elsevier Academic Press, 429–43.
James AP, Geijskes RJ, Dale JL, Harding RM, 2011. Molecular characterization of six
badnavirus species associated with leaf streak disease of banana in East Africa.
Annals of Applied Biology 158, 346–53.
Javer-Higginson E, Acina-Mambole I, González JE et al., 2014. Occurrence,
prevalence and molecular diversity of banana streak viruses in Cuba. European
Journal of Plant Pathology 138, 157–66.
Kearse M, Moir R, Wilson A et al., 2012. Geneious Basic: An integrated and
extendable desktop software platform for the organization and analysis of
sequence data. Bioinformatics 28, 1647–9.
Kenyon L, Lebas BSM, Seal SE, 2008. Yams (Dioscorea spp.) from the South Pacific
170
Islands contain many novel badnaviruses: implications for international
movement of yam germplasm. Archives of Virology 153, 877–89.
Kenyon L, Shoyinka S a, Hughes J d’A., Odu BO, 2001. An overview of viruses
infecting Dioscorea yams in sub-Saharan Africa. In: Plant Virology in Sub-
Saharan Africa. Proceedings of a Conference Organized by IITA. Ibadan,
Nigeria: IITA
Kleinow T, Nischang M, Beck A et al., 2009. Three C-terminal phosphorylation sites
in the Abutilon mosaic virus movement protein affect symptom development and
viral DNA accumulation. Virology 390, 89–101.
Kumar S, Stecher G, Tamura K, 2016. MEGA7: molecular evolutionary genetics
analysis version 7.0 for bigger datasets. Molecular biology and evolution 33,
1870–4.
Laney AG, Hassan M, Tzanetakis IE, 2012. An integrated badnavirus is prevalent in
fig germplasm. Phytopathology 102, 1182–9.
Larkin MA, Blackshields G, Brown NP et al., 2007. Clustal W and Clustal X version
2.0. Bioinformatics 23, 2947–8.
Lebot V, 2009. Tropical Root and Tuber Crops: Cassava, Sweet Potato, Yams and
Aroids. France: Paris University.
Medberry SL, Olszewski NE, 1993. Identification of cis elements involved in
Commelina yellow mottle virus promoter activity. The Plant Journal 3, 619–26.
Muhire BM, Varsani A, Martin DP, 2014. SDT: A virus classification tool based on
pairwise sequence alignment and identity calculation. PLoS ONE 9, e108277.
Muller E, Ravel S, Agret C et al., 2018. Next generation sequencing elucidates cacao
badnavirus diversity and reveals the existence of more than ten viral species.
Virus Research 244, 235–51.
171
Odu BO, Hughes JDAA, Asiedu R, Ng NQ, Shoyinka SA, Oladiran OA, 2004.
Responses of white yam (Dioscorea rotundata) cultivars to inoculation with three
viruses. Plant Pathology 53, 141–147.
Orkwor GC, 1998. The importance of yams. In: Orkwor GC,, Asiedu R,, Ekanayake
IJ, eds. Food Yams. Advances in Research. Nigeria: IITA (Ibadan) and NRCRI
(Umudike), 1–26.
Phillips S, Briddon RW, Brunt AA, Hull R, 1999. The partial characterization of a
badnavirus infecting the greater Asiatic or water yam (Dioscorea alata). Journal
of Phytopathology 147, 265–9.
Seal S, Turaki A, Muller E et al., 2014. The prevalence of badnaviruses in West
African yams (Dioscorea cayenensis-rotundata) and evidence of endogenous
pararetrovirus sequences in their genomes. Virus Research 186, 144–54.
Sharma SK, Vignesh Kumar P, Geetanjali AS, Pun KB, Baranwal VK, 2015.
Subpopulation level variation of banana streak viruses in India and common
evolution of banana and sugarcane badnaviruses. Virus Genes 50, 450–65.
Sharma SK, Vignesh Kumar P, Poswal R et al., 2014. Occurrence and distribution of
banana streak disease and standardization of a reliable detection procedure for
routine indexing of banana streak viruses in India. Scientia Horticulturae 179,
277–83.
Sukal A, Kidanemariam D, Dale J, James A, 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.
Sukal A, Kidanemariam D, Harding R, Dale J, James A, 2018. An improved
degenerate-primed rolling circle amplification and next-generation sequencing
172
approach for the detection and characterization of badnaviruses. Manuscript in
preparation.
Sukal A, Kidanemariam D, Harding R, Dale J, 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.
Sukal AC, Taylor M, Tuia VS, 2015. Viruses and their impact on the utilization of
plant genetic resources in the Pacific. Acta Horticulturae 1101, 127–32.
Tamiru M, Natsume S, Takagi H et al., 2017. Genome sequencing of the staple food
crop white Guinea yam enables the development of a molecular marker for sex
determination. BMC Biology 15, 86.
Umber M, Filloux D, Muller E et al., 2014. The genome of African yam (Dioscorea
cayenensis-rotundata complex ) hosts endogenous sequences from four distinct
badnavirus species. Molecular Plant Pathology 15, 790-801.
Wambulwa MC, 2012. Rolling circle amplification is more sensitive than PCR and
serology-based methods in detection of banana streak virus in Musa germplasm.
American Journal of Plant Sciences 03, 1581–7.
Wambulwa MC, Wachira FN, Karanja LS, Kiarie SM, Muturi SM, 2013. The
influence of host and pathogen genotypes on symptom severity in banana streak
disease. African Journal of Biotechnology 12, 27–31.
Yang IC, Hafner GJ, Dale JL, Harding RM, 2003. Genomic characterization of taro
bacilliform virus. Archives of Virology 148, 937–49.
173
Chapter 7
General Discussion
Food production per capita has decreased in nearly all the Pacific Island
countries (PICs) over the last decade, even in countries with little population growth
(SPC and CSIRO, 2011). With the imminent threat to food security from climate
change, agricultural production needs to be increased to maintain food security in the
PICs. Further, agricultural production is an important source of employment and
income for the PICs. Yam (mainly Dioscorea alata and D. esculenta) is one of the
major staples and ranks among the top root crops along with cassava (Manihot
esculenta) and aroids (Colocasia spp. and Xanthosoma spp.) (Elevitch and Love, 2011;
SPC and CSIRO, 2011). In the PICs, yam is grown together with other Pacific staple
crops, such as taro, coconut, breadfruit, banana and mango, to ensure adequate and
reliable production as part of the coping mechanisms to accommodate for the high
climate variability that exists in the region (Thaman, 2007). Wild yams (Dioscorea
spp.) play an important role during adverse climatic events, such as drought and
cyclones, when other staples are in short supply (Risimeri, 2001). Yam is mainly
consumed locally where it is grown, but it has become an export commodity for some
PICs, such as Fiji and Tonga, and has the potential to be developed further. Despite
having great potential, the development of yam has remained slow mainly due to the
low genetic diversity of cultivars in some of the PICs. This constraint restricts efforts
to select for desired traits such as quality, shape and size of tubers, which are conducive
for supply to export markets, as well as nutritional benefits and resistance to pests and
diseases. The lack of genetic diversity coupled with the rare and often male-dominated
flowering of the Pacific yam (D. alata) also prevents crop improvement through plant
breeding activities. Access to yam genetic resources from both within the Pacific
174
region and from outside the region is vital to inject the much needed genetic base to
support further improvement of Pacific yams and to support effective utilization. The
raw material to drive the desired improvement of yams in the Pacific exists within
globally recognized genetic repositories, namely SPC-CePaCT (Suva, Fiji) and IITA
(Nigeria), as accessions maintained in tissue culture and are available for exchange
under the Multilateral System of the International Treaty on Plant Genetic Resources
for Food and Agriculture (ITPGRFA). However, the exchange of yam germplasm has
remained limited, mainly due to the unavailability of diagnostic protocols to test
germplasm collections for viruses, particularly badnaviruses.
Over the last decade significant efforts have been directed towards improving
the understanding and knowledge of badnaviruses infecting yams and improving
diagnostic assays for their detection, however, knowledge on the diversity of
badnaviruses in the Pacific has been limited to the work done by Kenyon et al. (2008).
This group used a PCR-based technique to characterize the diversity of yam
badnaviruses, generally referred to as Dioscorea bacilliform viruses or DBVs, present
in the Pacific and reported 11 putative badnavirus species (K1-K11) of DBVs in yam
accessions. However, since PCR-based testing can amplify integrated sequences, the
episomal nature of these 11 groups was questionable. Kenyon et al. (2008) also
identified two sequence groups (K12-K13) that clustered distantly from other DBV
sequence groups and outside of, but placed between, members of the genera
Badnavirus and Tungrovirus. This raised further questions as to whether additional
viral groups distantly related to genera Badnavirus and Tungrovirus were prevalent in
Pacific yams. The results presented in this thesis resolve some of the questions left
unanswered by previous studies on yam badnaviruses in the Pacific. Further, studies
on the diversity of DBVs prevalent in Pacific yam germplasm conserved at SPC-
175
CePaCT and the development of protocols to support the testing of yam germplasm
for DBVs were done to enable the safe exchange of yam germplasm without risk of
disseminating DBVs.
7.1. Dioscorea bacilliform virus (DBV)
At the start of the present study the existence of episomal DBVs in Pacific yams
was unknown. In addition, only two episomal DBV species, namely Dioscorea
bacilliform SN virus (DBSNV) and Dioscorea bacilliform AL virus (DBALV), had
been characterized from the African and Caribbean regions, respectively. Concurrent
with the work described in this thesis, additional DBV species were characterized from
the African and Caribbean region, including Dioscorea bacilliform RT virus 1
(DBRTV1), and Dioscorea bacilliform RT virus 2 (DBRTV2) (Bömer et al., 2016), as
well as Dioscorea bacilliform RT virus 3 (DBRTV3) (Bömer et al., 2018) and
Dioscorea bacilliform TR virus (DBTRV) (Umber et al., 2017). In the present study,
a total of 224 out of 283 yam germplasm conserved at SPC-CePaCT were screened
using rolling circle amplification (RCA). Using a combination of Sanger and next-
generation-sequencing (NGS), a total of 24 complete DBV genomes representing four
different species groups, namely K1 or Dioscorea bacilliform ES virus (DBESV), K3
or Dioscorea bacilliform AL virus 2 (DBALV2), K8 or DBALV and T14 or Dioscorea
bacilliform RT virus 2 (DBRTV2), were identified in the Pacific. Two of the four
DBVs, namely DBALV2 and DBESV, were determined to be novel species and shown
to be restricted to D. alata from PNG and D. esculenta from Fiji, respectively.
Similarly, DBRTV2 was only identified from Samoa and DBALV was identified from
Tonga and Vanuatu. None of the accessions tested from New Caledonia or Federated
States of Micronesia (FSM) were found to be infected with DBVs. DBALV was the
176
only virus found to be present in two countries, Vanuatu and Tonga, while, DBALV2,
DBESV and DBRTV2 were found to be restricted to single countries, namely PNG,
Fiji and Samoa, respectively.
The results of this study considerably expands the knowledge of episomal DBVs
in the Pacific region with four known species and 24 complete genomes now
characterized. This study, together with the efforts of other researchers over the last
four years (Bömer et al., 2016, 2018; Umber et al., 2017), has increased the knowledge
of episomal DBVs from two to seven species. A greater understanding of the diversity
of DBVs will enable the development of reliable diagnostic protocols to support
screening of germplasm collections for safe germplasm exchange. As illustrated from
this study, some DBVs appear to be endemic to certain PICs, reinforcing the need for
effective screening measures to ensure that they are not distributed throughout the
region.
7.2. Dioscorea nummularia-associated virus (DNUaV)
In addition to describing the diversity of DBVs in the Pacific, this study has
characterized an additional virus sequence that is phylogenetically distinct from
badnaviruses, but clearly belongs within the family Caulimoviridae. Currently, the
family Caulimoviridae contains eight recognized genera, namely Badnavirus
Caulimoviridae, Cavemovirus, Petuvirus, Rosadnavirus, Soymovirus, Solendovirus,
Tungrovirus. Kenyon et al. (2008) and Turaki et al. (2017), using universal badnavirus
(BadnaFP/RP) primers (Yang et al., 2003), obtained partial RT/RNase H-coding
sequences which, following sequence comparisons, were subsequently delineated into
four distinct groups, namely K12, K13, T16 and T17, respectively. These sequences
were found to represent distinct groups with very low nucleotide identity to DBV
177
sequences and other badnavirus sequences. Nevertheless, during phylogenetic analysis
the sequences clustered outside of, but between, the genera Badnavirus and
Tungrovirus. However, none of the studies were able to determine if groups K12, K13,
T16 and T17 were derived from integrated or episomal sequences, or if they
represented new genera within the family Caulimoviridae. In the current study, RCA
was used to amplify, clone and sequence the complete genome of an additional
sequence group, namely Dioscorea nummularia-associated virus (DNUaV) which,
according to the demarcation criteria set out by ICTV for species delineation within
current genera of the family Caulimoviridae, was determined to be a novel sequence
group. During phylogenetic analysis, DNUaV clustered together with K12 and T16
providing strong evidence that these sequences could also be of episomal origin.
Genome size, organization and the presence of conserved amino acid domains of
DNUaV are characteristic of members of the family Caulimoviridae. However,
nucleotide sequence similarity and phylogenetic analysis suggests that DNUaV could
be a distinct novel member of the family and may represent a new genus within the
Caulimoviridae family. Despite the strong evidence presented for the existence of
episomal DNUaV sequence, further studies on virus transmission, virion morphology
and whether infected plants contain inclusion bodies typical of members of the genus
Caulimovirus are required before the taxonomic status of DNUaV can be fully
resolved. The occurrence of DNUaV-like virus in other yam species and countries
also needs further investigation.
178
7.3. Development of diagnostic protocols
Increased knowledge on the diversity of episomal DBV’s and related viruses
present in the Pacific and other regions opens opportunities for the development of
diagnostic protocols which will support the indexing and safe distribution/exchange of
much needed genetic diversity. This study contributes to this global effort. As alluded
to earlier, detection of DBVs presents a particular challenge mainly because of lack of
knowledge on episomal sequences, the presence of host integrated sequences and the
heterogeneous nature, both genetically and serologically, of DBV.
In addition to the identification and characterization of DBVs, this study also
investigated techniques for the diagnosis of DBVs. Initially, PCR protocols were
developed for the detection of DBALV2, DBESV and DBRTV2, while DNUaV-
specific primers for PCR detection of DNUaV were also designed. Although these
PCR protocols successfully detected the isolates characterized in this study, their
ability to detect the breadth of DBV isolates and to differentiate between episomal and
integrated sequences was unknown. Due to these potential shortcomings, RCA-based
protocols were considered more appropriate. James et al. (2011a) showed the utility of
RCA for the detection of badnaviruses from banana using random-primed RCA with
addition of specific badnavirus primers (primer-spiked RP-RCA). Based on this work
many authors have successfully used RCA for the diagnosis of episomal badnavirus
infections in different hosts, including banana (Baranwal et al., 2014; Carnelossi et al.,
2014; James et al., 2011b; Javer-Higginson et al., 2014; Wambulwa et al., 2012;
Wambulwa et al., 2013; Sharma et al., 2014, 2015), cacao (Chingandu et al., 2017b,
2017a; Muller et al., 2018), fig (Laney et al., 2012), mulberry (Chiumenti et al., 2016),
Rubus spp. (Diaz-Lara et al., 2015) and yam (Bömer et al., 2018, 2016; Sukal et al.,
2017; Umber et al., 2014). However, since RCA amplification of DBVs from yams is
179
not always reliable and has been shown to be unsuitable for DBV indexing (Bömer et
al., 2016; Umber et al., 2014), this study sought to optimize the existing protocols.
During the initial DBV characterization studies, it was found that sequence-
independent RCA using the TempliPhi™ kit (GE Healthcare) yielded inconsistent
results, even with the addition of virus-specific primers similar to the strategy
described by James et al. (2011a). Bömer et al. (2016) found that the inclusion of a
purification step, following total nucleic acid extraction, with Tip-100G columns
improved the efficiency of RCA. However, Bömer et al. (2016) provided experimental
evidence that, even with total nucleic acid purification, RCA still resulted in high
levels of false negatives and could amplify linear templates. The nature of many
commercial RCA kits, such as TempliPhi™, restricts the optimization of the reaction
components. Therefore, a strategy to optimize RCA protocols by using directed-RCA
(D-RCA) or specific-primed-RCA (SP-RCA), which preferentially amplify episomal
badnavirus DNA, were compared to previously published RCA protocols such as
random-primed approach (RP-RCA) and primer-spiked RP-RCA. Using NGS to
analyse the reaction products, D-RCA and SP-RCA were found to be approximately
80-fold more efficient in DBV amplification than the TempliPhi kit protocols using
the manufacturer’s procedures. During the optimization of the RCA protocols, an
incubation temperature of 36°C for the TempliPhi RCA was also found to result in
greater target amplification than the manufacturer recommended 30°C for the
TempliPhi RCA protocols. When the D-RCA and SP-RCA protocols were assessed
using a range of different badnaviruses from different host plant species, all viruses
were successfully amplified from all hosts tested. Further, D-RCA was shown to be
the most consistent and efficient protocol for the amplification of DBVs (DBALV,
DBALV2, DBESV, DBRTV2 and DBRTV3), BSV (BSMYV, BSGFV and BSOLV),
180
SCBIMV and TaBV, and performed comparatively better than the RP-RCA or primer-
spiked RP-RCA approach using the TempliPhi kit. While SP-RCA was not as robust
as D-RCA, it nonetheless performed as well, if not better, than the TempliPhi kit
protocols for badnavirus detection. Since the SP-RCA protocol only utilizes
badnavirus genus-specific primers, its efficiency can be further improved with the
inclusion of additional primers.
The D-RCA and SP-RCA protocols successfully amplified all DBV species
from the Pacific. Although DBV species from regions outside the Pacific were not able
to be accessed to validate the described protocols, in silico analysis revealed that,
theoretically, they can be used to detect a broad range of DBV species. In the future,
it is hoped that, through collaborative efforts with researchers in other countries, the
protocols described here can be evaluated with other DBV species and an optimized
global DBV diagnostic protocol developed. However, this study has still shown that
RCA works well for the DBV species present in the Pacific and that following RCA
restriction analysis (using endonuclease such as EcoRI, KpnI, SphI and StuI) the
presence or absence of DBVs can be successfully determined. Although further effort
is needed to be able to fully identify species of DBV present, the presence/absence of
DBV is sufficient to enable germplasm screening and address the issue of safe
dissemination.
This study has also shown that RCA, in combination with NGS, can be an
effective tool for virus detection and characterization. Although the costs of NGS may
currently preclude its routine use, with the improvement of the technology and likely
decrease in costs of NGS, it may be an option in the future. Further research into
identification of sequence variants and quasispecies present in infected samples, and
181
the relationship to differences in restriction digest patterns will strengthen
interpretation of RCA restriction results.
7.4. Conclusions
Yam (Dioscorea spp.) is an important food staple in the Pacific with great
potential for increasing food security and commercial exploitation. However, the
agronomical challenges with its production have limited its utilization. Pacific
germplasm collections conserved at SPC-CePaCT and other important collections,
such as those conserved by IITA, hold germplasm that are integral to overcoming these
agronomical challenges. However, access to this germplasm has remained limited due
to concerns over introduction of new viral diseases with the germplasm. DBVs are of
great concern mainly because of a lack of reliable diagnostic tests which prevents
routine indexing for safe exchange. The RCA protocols optimized in this study have
huge potential for episomal virus characterization when coupled with restriction
analysis alone and/or Sanger sequencing and/or direct sequencing of the RCA products
via NGS. This provides an avenue to virus index yam germplasm and certify them free
of episomal DBVs. To date there is no clear evidence of activation of endogenous
DBV-like sequences, however, the possibility still exists. Therefore, RCA protocols
proposed in this study, coupled with either restriction analysis, Sanger Sequencing and
NGS alone or a combination, provides the best possible option for the detection and
characterization of episomal DBVs.
Since the present study was not able to obtain DBV species from regions other
than the Pacific, a global collaborative effort is warranted, firstly to determine the
DBVs that are present in the different yam growing regions, mainly the African and
Caribbean regions and secondly, to utilize the methods described in this study to
182
evaluate their efficacy as a universal diagnostic tool for DBV. The RCA methods
described coupled with NGS could be deployed to other regions to characterize the
yam DBVs present. Sequence information on DBVs from the different regions will
help in the development of universal primers which could be used in RCA methods
for DBV diagnosis to support transboundary safe yam germplasm exchange.
For the immediate future, techniques described in this study will be used at SPC-
CePaCT under an ACIAR funded integrated crop management (ICM) project titled:
Integrated crop management strategies for root and tuber crops: strengthening
national and regional capacities in Papua New Guinea, Fiji, Samoa, Solomon Islands
and Tonga (Project number: HORT/2010/065), to screen the Pacific yam germplasm
collections for DBV and other viruses including those in the families Alphaflexiviridae
(genus Potexvirus), Betaflexiviridae (genus Carlavirus), Bromoviridae (genus
Cucumovirus), Caulimoviridae (genus Badnavirus), Potyviridae (genus Macluravirus
and Potyvirus), Secoviridae (genus Comovirus and Fabavirus) and Tombusviridae
(genus Aureusvirus). Accessions found to be free from these viruses will then be made
available for distribution. The virus testing results will enable the yam germplasm to
meet quarantine requirements enabling access by the different Pacific Island
Countries.
Access to yam germplasm by the countries will enable the evaluation of the
different cultivars available at SPC-CePaCT for desired agronomical traits.
Identification of cultivars with desirable agronomical traits, such as anthracnose
resistance, will enable local farmers to increase subsistence production and
commercial exploitation. Countries such as Fiji, Samoa and Tonga, through evaluation
stand to benefit from identification of cultivars with tuber morphology conducive to
export. Some yam cultivars (especially the lesser yams D. esculenta) have also been
183
identified as drought tolerant. Some have been identified suitable for atoll islands, such
as Viwa Island in Fiji, and can be distributed to enhance tolerance against drought thus
contributing towards resilience against climate change (SPC data, unpublished).
The unique germplasm conserved at SPC-CePaCT has the promise of desired
agronomical traits that will promote informed utilization and exploitation of yam
genetic material to build food and nutritional security in the Pacific and also support
resilience against climate change.
184
7.5. References
Baranwal VK, Sharma SK, Khurana D, Verma R, 2014. Sequence analysis of shorter
than genome length episomal banana streak OL virus like sequences isolated from
banana in India. Virus Genes 48, 120–127.
Bömer, M., Rathnayake, A.I., Visendi, P., Silva, G., Seal, S.E., 2018. Complete
genome sequence of a new member of the genus Badnavirus, Dioscorea
bacilliform RT virus 3, reveals the first evidence of recombination in yam
badnaviruses. Arch. Virol. 163, 533–538.
Bömer, M., Turaki, A., Silva, G., Kumar, P., Seal, S., 2016. A sequence-independent
strategy for amplification and characterization of episomal badnavirus sequences
reveals three previously uncharacterized yam badnaviruses. Viruses 8, 188.
Carnelossi, P.R., Bijora, T., Facco, C.U., Silva, J.M., Picoli, M.H.S., Souto, E.R.,
Oliveira, F.T. De, 2014. Episomal detection of banana streak OL virus in single
and mixed infection with Cucumber mosaic virus in banana “Nanicão Jangada.”
Trop. Plant Pathol. 39, 342–346.
Chingandu, N., Kouakou, K., Aka, R., Ameyaw, G., Gutierrez, O.A., Herrmann, H.-
W., Brown, J.K., 2017a. The proposed new species, cacao red vein virus, and
three previously recognized badnavirus species are associated with cacao swollen
shoot disease. Virol. J. 14, 199.
Chingandu, N., Zia-ur-rehman, M., Sreenivasan, T.N., Surujdeo-Maharaj, S.,
Umaharan, P., Gutierrez, O.A., Brown, J.K., Thyail, M.Z., Zia-ur-rehman, M.,
Sreenivasan, T.N., Surujdeo-Maharaj, S., Umaharan, P., Gutierrez, O.A., Brown,
J.K., Thyail, M.Z., 2017b. Molecular characterization of previously elusive
badnaviruses associated with symptomatic cacao in the New World. Arch. Virol.
162, 1363–1371.
185
Chiumenti, M., Morelli, M., De Stradis, A., Elbeaino, T., Stavolone, L., Minafra, A.,
2016. Unusual genomic features of a badnavirus infecting mulberry. J. Gen.
Virol. 97, 3073–3087.
Diaz-Lara, A., Mosier, N.J., Keller, K.E., Martin, R.R., 2015. A variant of Rubus
yellow net virus with altered genomic organization. Virus Genes 50, 104–110.
Elevitch, B.C.R., Love, K., 2011. Farm and Forestry Production and Marketing
Profiles: Highlighting value-added strategies., in: Elevitch, C.R. (Ed.), Specialty
Crops for Pacific Island Agroforestry. Permanent Agriculture Resources (PAR),
Holualoa, Hawai‘i., p. 14.
James, A.P., Geijskes, R.J., Dale, J.L., A., Harding, R.M., 2011a. Development of a
novel rolling-circle amplification technique to detect banana streak virus that also
discriminates between integrated and episomal virus sequences. Plant Dis. 95,
57–62.
James, A.P., Geijskes, R.J., Dale, J.L., Harding, R.M., 2011b. Molecular
characterization of six badnavirus species associated with leaf streak disease of
banana in East Africa. Ann. Appl. Biol. 158, 346–353.
Javer-Higginson, E., Acina-Mambole, I., González, J.E., Font, C., González, G.,
Echemendía, A.L., Muller, E., Teycheney, P.Y., 2014. Occurrence, prevalence
and molecular diversity of banana streak viruses in Cuba. Eur. J. Plant Pathol.
138, 157–166.
Kenyon, L., Lebas, B.S.M., Seal, S.E., 2008. Yams (Dioscorea spp.) from the South
Pacific Islands contain many novel badnaviruses: implications for international
movement of yam germplasm. Arch. Virol. 153, 877–889.
Laney, A.G., Hassan, M., Tzanetakis, I.E., 2012. An integrated badnavirus is prevalent
in fig germplasm. Phytopathology 102, 1182–1189.
186
Muller, E., Ravel, S., Agret, C., Abrokwah, F., Dzahini-Obiatey, H., Galyuon, I.,
Kouakou, K., Jeyaseelan, E.C., Allainguillaume, J., Wetten, A., 2018. Next
generation sequencing elucidates cacao badnavirus diversity and reveals the
existence of more than ten viral species. Virus Res. 244, 235–251.
Risimeri, J.B., 2001. Yams and food security in the lowlands of PNG, in: Food
Security for Papua New Guinea. Australian Centre for International Agricultural
Research, Canberra.
Sharma, S.K., Vignesh Kumar, P., Geetanjali, A.S., Pun, K.B., Baranwal, V.K., 2015.
Subpopulation level variation of banana streak viruses in India and common
evolution of banana and sugarcane badnaviruses. Virus Genes 50, 450–465.
Sharma, S.K., Vignesh Kumar, P., Poswal, R., Rai, R., Swapna Geetanjali, A., Prabha,
K., Jain, R.K., Baranwal, V.K., 2014. Occurrence and distribution of banana
streak disease and standardization of a reliable detection procedure for routine
indexing of banana streak viruses in India. Sci. Hortic. 179, 277–283.
SPC and CSIRO, 2011. Food security in the Pacific and East Timor and its
vulnerability to climate change. Pacific Community (SPC), Noumea.
Sukal, A., Kidanemariam, D., Dale, J., James, A., 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 Res. 238,
29–34.
Thaman, R.R., 2007. Restoring the Pacific Islands’ rich agricultural traditions: an
urgent priority. Pacific Ecol. 15, 51–57.
Turaki, A.A., Bömer, M., Silva, G., Lava Kumar, P., Seal, S.E., 2017. PCR-DGGE
analysis: Unravelling complex mixtures of badnavirus sequences present in yam
germplasm. Viruses 9, 181.
187
Umber, M., Filloux, D., Muller, E., Laboureau, N., Galzi, S., Roumagnac, P., Iskra-
Caruana, M.-L., Pavis, C., Teycheney, P.-Y., Seal, S.E., 2014. The genome of
African yam (Dioscorea cayenensis-rotundata complex) hosts endogenous
sequences from four distinct badnavirus species. Mol. Plant Pathol. 15, 790–801.
Umber, M., Gomez, R.M., Gélabale, S., Bonheur, L., Pavis, C., Teycheney, P.Y.,
2017. The genome sequence of Dioscorea bacilliform TR virus, a member of the
genus Badnavirus infecting Dioscorea spp., sheds light on the possible function
of endogenous Dioscorea bacilliform viruses. Arch. Virol. 162, 517–521.
Wambulwa, M.C., 2012. Rolling Circle Amplification Is More Sensitive than PCR
and Serology-Based Methods in Detection of Banana streak virus in Musa
Germplasm. Am. J. Plant Sci. 03, 1581–1587.
Wambulwa, M.C., Wachira, F.N., Karanja, L.S., Kiarie, S.M., Muturi, S.M., 2013.
The influence of host and pathogen genotypes on symptom severity in banana
streak disease. African J. Biotechnol. 12, 27–31.
Yang I.C., Hafner G.J., Dale J.L., Harding R.M., 2003. Genomic characterization of
taro bacilliform virus. Arch. Virol. 148, 937–49.