ABSTRACT
Ralstonia solanacearum is a genetically diverse and geographically widespread plant
pathogen. It has a wide host range and is a significant pathogen of potato, causing
brown rot. Brown rot is caused by a distinct, closely-related, intraspecific group: race
3, biovar 2A. In Europe, infection of potato crops with brown rot primarily occurs via
irrigation with contaminated surface water. Brown rot has never been found in
Scottish potatoes though the bacterium has been found previously in one Scottish
river system, the River Tay, both in water samples and on its secondary host,
bittersweet (Solanum dulcamara), growing on the river banks. A molecular straintyping method principally used in clinical microbiology, multi-locus sequence
typing (MLST), was used to study genetic variation within a collection of 106 R.
solanacearum isolates, principally race 3 biovar 2A isolates from potato, S. dulcamara
and contaminated water sources. Twenty-seven isolates from contaminated water and S.
dulcamara from Scotland and other isolates from diverse geographic locations, from a
variety of diseased plants and the environment, were resolved into 16 sequence types. A
subsequent follow-up to the first experiment was carried out by looking at more
variable genes within the race 3, biovar 2A genome and again similar or identical
relationships were uncovered. All Scottish isolates were found to be identical and
similar to most race 3 biovar 2A isolates tested. Analysis of Variable Number
Tandem Repeats (VNTRs) confirmed this observation. After sequencing one of the
tandem repeat regions, the results strongly suggest that contamination of the River
Tay in Scotland occurred as a single or limited event, since the Scottish isolates had a
unique tandem repeat pattern different from the patterns observed for the rest of the
race 3 biovar 2A isolates and strains studied. This suggests that the Scottish isolates
are clonal and the contamination is a single event and not a multiple contamination.
i
ACKNOWLEDMENTS
First and foremost I offer my sincerest gratitude to my supervisor, Dr Gerry Saddler,
Science and Advice for Scottish Agriculture (SASA), who has supported me
throughout my PhD with his patience and knowledge whilst allowing me the room to
work in my own way. I attribute the level of my PhD to his encouragement and effort
and without him this thesis would not have been completed or written. I could not
wish for a better or friendlier supervisor. I owe my deepest gratitude to Dr Wilf
Mitchell (Heriot-Watt University) for his invaluable guidance and support throughout
the PhD.
I am grateful to Dr Pieter van de Graaf, Dr Alex Reid, Mr Vince Mulholland and Dr
Rosalind McHugh for all their guidance, technical help, support and encouragement
throughout my studies.
I wish to thank all the staff at the Diagnostic and Molecular Biology section for their
support and encouragement throughout my studies. My sincere regards also goes to
everyone at SASA whose support made moving to Edinburgh an exciting experience.
I am grateful to SASA for providing me the support and equipment I have needed to
produce and complete my thesis and the Scottish Executive (Flexible Fund) for
funding my project.
Finally, I thank my parents for supporting me throughout all my studies at University
and my wife and sisters for all their encouragement.
It is an honour for me to have under taken my PhD at SASA and I thoroughly
enjoyed my experience.
ii
CONTENTS
1.
Introduction…………………………………………………………
1
1.1
Ralstonia solanacearum……………………………………………...
1
1.1.1
Biology...……………………………………………………………..
1
1.1.2
Infection mechanism…………………………………………………
2
1.1.3
Geographic distribution………………………………………………
3
1.1.4
Disease status………………………………………………………...
4
1.2
Ralstonia solanacearum race 3 biovar 2A…………………………...
4
1.2.1
Biology……………………………………………………………….
4
1.2.2
Infection mechanism…………………………………………………
5
1.2.3
Geographic distribution………………………………………………
7
1.2.4
Disease status………………………………………………………...
9
1.3
Detection of Ralstonia solanacearum……………………………….
10
1.3.1
Selective isolation……………………………………………………
10
1.3.2
PCR…………………………………………………………………..
11
1.3.2.1
Conventional PCR……………………………………………………
12
1.3.2.2
Real-time PCR……………………………………………………….
12
1.3.3
Immunofluorescence (IF)…………………………………………….
14
1.3.4
Enzyme Linked Immunosorbent Assay (ELISA)…………………….
15
1.4
Characterisation of Ralstonia solanacearum…………………………
16
1.4.1
Biochemical tests……………………………………………………..
16
1.4.2
BIOLOG microbial characterisation…………………………………
16
1.4.3
Molecular methods…………………………………………………...
17
1.4.3.1
Fingerprinting………………………………………………………...
17
1.4.3.1.1
PCR-Restriction Fragment Length Polymorphism (RFLP)………….
18
1.4.3.1.2
PFGE…………………………………………………………………
19
1.4.3.1.3
Amplified fragment length polymorphism analysis (AFLP)…………
20
1.4.3.1.4
Repetitive sequences polymerase chain reaction…………………….
20
1.4.3.2
Sequenced based methods……………………………………………
22
1.4.3.2.1
6S rDNA sequencing…………………………………………………
22
1.4.3.2.2
Sequencing of the endoglucanase gene………………………………
23
iii
1.4.3.2.3
Phylotyping scheme for R. solanacearum……………………………
23
1.4.3.2.4
Whole bacterium genome sequencing………………………………..
24
1.4.3.3
Multi-locus sequence typing (MLST)…………..................................
25
1.4.3.3.1
Introduction and overview……………………………………………
25
1.4.3.3.2
Examples of existing databases………………………………………
28
1.4.3.3.3
MLST scheme for Neisseria meningitidis……
28
1.4.3.3.4
MLST scheme for Burkholderia thailandensis, B. pseudomallei & B.
mallei…………………………………………………………………
29
1.4.3.3.5
MLST scheme for Bacillus species…………………………………..
30
1.4.3.3.6
MLST scheme for Campylobacter jejuni…………………………….
30
1.4.3.3.7
MLST scheme for Escherichia coli…………………………………..
31
1.4.3.3.8
MLST scheme for Candida albicans……….......................................
32
1.4.4
Other methods for analysing population……………………………..
32
1.4.4.1
Single Nucleotide Polymorphisms (SNPs)…………………………..
33
1.4.4.2
Variable Number Tandem Repeats (VNTRs)………………………..
34
1.5
Potato industry………………………………………………………..
36
1.5.1
Potato industry in Scotland…………………………………………...
37
1.6
Brown Rot Project……………………………………………………
38
1.6.1
Aims of the brown rot project ………………………………………..
38
1.6.2
Aims of this PhD project……………………………………………..
39
2.
Materials and Method………………………………………………
41
2.1
Properties of bacterial isolates………………………………………..
41
2.2
Cultivation of Ralstonia solanacearum…..…………………………..
41
2.3
Extraction of chromosomal DNA…………………………………….
42
2.4
Multilocus Sequence Typing (MLST)………………………………..
43
2.4.1
MLST-1………………………………………………………………
43
2.4.1.1
Primers used in MLST-1……………………………………………..
44
2.4.1.2
Initial fragment amplification for MLST…………………………….
45
2.4.1.3
Purification of sequencing template………………………………….
47
2.4.1.4
Cycle sequencing reactions of PCR products………………………...
48
2.4.1.5
Purification of products after cycle sequencing……………...............
50
2.4.1.6
DNA sequencing……………………………………………………..
51
2.4.1.7
Processing of sequence data………………………………………….
51
iv
2.4.2
Data analysis………………………………………………………….
52
2.4.3
MLST-2………………………………………………………………
52
2.4.3.1
Primers used in MLST-2……………………………………………..
54
2.4.3.2
Processing of isolates……………………….......................................
56
2.5
Variable number tandem repeats (VNTRs)…………………………..
56
2.5.1
Marker design for VNTRs…………………………………………....
57
2.5.1.1
Tetra-nucleotide repeats……………………………………...............
57
2.5.1.2
Di-nucleotide repeats…………………………………………............
58
2.5.1.3
Mono-nucleotide repeats……………………………………..............
58
2.5.2
Polymerase chain reaction amplifications for VNTRs……….............
59
2.5.3
Analysis of PCR products……………………………………............
61
2.6
Sequencing VNTRs region…………………………………………...
62
2.7
Investigation of the endoglucanase gene……………………………..
63
2.8
BOX PCR…………………………………………………………….
63
2.8.1
BOX PCR methodology……………………………………...............
63
2.8.2
BOX PCR Analysis…………………………………………..............
65
2.9
TaqMan® assay……………………………………………………...
66
2.9.1
TaqMan® methodology……………………………………..............
66
2.9.2
Post-PCR analysis…………………………………………................
68
2.10
Biovar testing………………………………………………………....
68
3.
Results………………………………………………………………..
70
3.1
Properties of bacterial isolates………………………………………..
70
3.2
Identification of selected isolates…………………………………….
70
3.2.1
Real-time PCR assay…………………………………………............
70
3.2.2
Biovar test…………………………………………………….............
71
3.3
The genetic diversity of R. solanacearum isolates and strains as
determined by molecular fingerprinting……………………………...
73
3.3.1
BOX-PCR analysis…………………………………………...............
73
3.4
The diversity of isolates and strains of R. solanacearum as
determined by sequence typing………………………………………
73
3.4.1
Multi-locus sequence typing-1 (MLST-1)…………………................
73
3.4.1.1
Loci (allele) selection………………………………………...............
74
3.4.1.2
Determination of primers/sequences…………………………............
74
v
3.4.1.3
Protein function………………………………………………............
76
3.4.1.4
MLST-1 results……………………………………………….............
77
3.4.1.5
Analysis of alleles…………………………………………….............
81
3.4.1.6
Frequently occurring alleles………………………………….............
81
3.4.2
MLST-2………………………………………………………............
83
3.4.2.1
New Loci (allele) selection…………………………………...............
83
3.4.2.2
Determination of primers/sequences…………………………............
83
3.4.2.3
Protein function……………………………………………................
85
3.4.2.4
MLST-2 results…………………………………………….................
86
3.4.2.6
Analysis of alleles………………………………………….................
90
3.5
Variable number tandem repeats (VNTRs)…………………………..
90
3.5.1
Mono-nucleotide repeats and di-nucleotide repeats………................
91
3.6
Sequencing of the endoglucanase gene……………………………....
93
3.7
Sequencing of the tandem repeat regions………………………….....
96
4.
Discussion……………………………………………………............
98
4.1
Studies on genetic diversity…………………………………………..
99
4.2
Phylotyping scheme..............................................................................
101
4.3
MLST………………………………………………………………...
102
4.4
New target selection.............................................................................
104
4.5
MLST schemes for plant pathogens………………………………….
106
4.6
Population genetics and MLST analysis……………………………..
107
4.7
Analysis of loci selected for the R. solanacearum MLST scheme…...
110
4.8
Endoglucanase sequencing…………………………………………...
112
4.9
Variable Number Tandem Repeats (VNTRs)......................................
114
4.10
Sequencing of the tandem repeat region UW551-R05789...................
118
4.11
General discussion................................................................................
120
4.12
Database for the Multi-Locus Sequence Typing of R. solanacearum..
123
4.13
Conclusions…………………………………………………………..
124
5.
References…………………………………………………………...
125
6.
Appendices…………………………………………………………..
151
vi
Appendix I
Details of Ralstonia solanacearum strains and isolates included
in this study
Appendix II
Verification of identity and biovar assignment of 106 isolates
and strains of R. solanacearum by quantitative PCR and biovar
tests
Appendix III
Diverse collection of isolates used in initial screening of
potential gene targets for MLST-1 and MLST-2
Appendix IV
Allele numbers and sequence types assigned to isolates and
strains of R. solanacearum analysed using MLST-1
Appendix V
Polymorphic sites of the seven genes included in MLST-1 from
the study of 106 R. solanacearum isolates and strains
Appendix VI
Allele numbers and sequence types assigned to isolates and
strains of R. solanacearum analysed using MLST-2
Appendix VII
Polymorphic sites of the seven genes included in MLST-2 from
the study of 106 R. solanacearum isolates and strains
Appendix VIII
Isolates selected for screening VNTRs markers
Appendix IX
Allele numbers and sequence types assigned to isolates and
strains of R. solanacearum analysed using VNTRs
Appendix X
Endoglucanase gene analysis of R. solanacearum isolates and
strains
Appendix XI
Variable Number Tandem Repeats (VNTRs) analysis of 88 R.
solanacearum isolates and strains
vii
LIST OF TABLES AND FIGURES
Tables
Table 1.1
Race classification of R. solanacearum, as described by Buddenhagen
et al. (1962); Buddenhagen (1986).
Table 1.2
Biovar classification of R. solanacearum, as described by Hayward
(1964)
Table 1.3
Geographic distribution
Table 1.4
Phylotype designation
Table 2.1
Genetic loci analysed in MLST, based on the R. solanacearum
GMI1000 genome
Table 2.2
Sequences and annealing temperatures of PCR primers used in MLST
Table 2.3
PCR constituents for the MLST housekeeping genes
Table 2.4
PCR cycle programme
Table 2.5
The sequencing reaction constituents, for the MLST housekeeping
genes
Table 2.6
Sequences of sequencing primers used in MLST-1
Table 2.7
Cycling programme for sequencing reactions
Table 2.8
MLST-2 scheme: loci targeted for initial screening, based on the R.
solanacearum UW551 genome
Table 2.9
Loci analysed in MLST-2, based on the R. solanacearum UW551
genome
Table 2.10
Sequences & annealing temperatures of PCR primers used in MLST-2
Table 2.11
Details of sequencing primers used in MLST-2
Table 2.12
Primer details for tetra-nucleotide repeats
Table 2.13
Primer details for di-nucleotide repeats
Table 2.14
Primer details for mono-nucleotide repeats
Table 2.15
Polymerase chain reaction constituents for VNTRs
Table 2.16
PCR cycle programme for VNTRs reaction
Table 2.17
Master-mix for VNTR analysis
Table 2.18
BOX PCR reaction constituents
Table 2.19
PCR cycle programme for BOX-PCR reactions
Table 2.20
Characteristics of primers and probes used in TaqMan assay®
viii
Table 2.21
Reaction constituents for the TaqMan® assay
Table 2.22
Cycle program for TaqMan® assay
Table 3.1
Summary of results: Quantitative PCR and Biovar tests
Table 3.2
Targets selected from the chromosome of R. solanacearum GMI1000
for use in MLST-1
Table 3.3
Gene products of the 7 component genes of the MLST-1 system
Table 3.4
Allele length, frequency and number of polymorphic sites of the
selected targets used in MLST-1
Table 3.5
Allele frequencies of the 106 isolates and strains of R. solanacearum
studied in MLST-1
Table 3.6
Genes selected from the chromosome and mega-plasmid of UW551
and their suitability as components of MLST-2
Table 3.7
MLST-2 products of the 7 component genes of the system, derived
from the complete genome sequence of the R. solanacearum UW551
genome
Table 3.8
Allele frequencies of the 106 isolates and strains of R. solanacearum
studied in MLST-2: Summary of STs
Table 3.9
Allele length, frequency and number of polymorphic sites of the
selected targets
Table 3.10
Scottish isolates and their VNTR types
Table 3.11
The sequence types recovered the endoglucanase gene analysis of R.
solanacearum isolates and strains
Table 3.12
VNTRs analysis of R. solanacearum isolates and strains
ix
Figures
Figure 1.1
Infected vascular bundles and surrounding tissue in a potato tuber
infected with R. solanacearum
Figure 1.2
Bacterial slime oozes from the tuber eyes as a result of infection with
R. solanacearum
Figure 1.3
Wilting of a potato plant infected with R. solanacearum
Figure 1.4
Bittersweet (Solanum dulcamara), a wild host of R. solanacearum
(SASA)
Figure 1.5
R. solanacearum World-wide distribution
Figure 1.6
R. solanacearum on SMSA agar (SASA). Colonies typically exhibit
purple centres with a white margin
Figure 1.7
MLST Methodologies
Figure 3.1
The distribution of the seven selected genes from MLST-1 on the
chromosome of GMI1000
Figure 3.2
MLST-1 phylogenetic trees generated by Maximum Likelihood
analysis of 106 isolates of R. solanacearum and the concatenated
sequence data from seven housekeeping genes
Figure 3.3
MLST-2 phylogenetic trees generated by Maximum Likelihood
analysis of 106 isolates of R. solanacearum from the
concatenated sequence data of seven genes from the chromosome and
mega-plasmid
Figure 3.4
Cluster analyses of the partial endoglucanase gene sequence of 104
isolates of R. solanacearum
x
LIST OF PRESENTATIONS AND POSTERS
Papers
1. Danial J., Saddler G.S. and van de Graaf P., 2006, Studies on the genetic
variation between isolates of the potato brown rot pathogen Ralstonia
solanacearum, Proceedings Crop Protection in Northern Britain, Dundee, 347352.
Presentations
1. Molecular Biology of Plant Pathogens XVI , 2005, Ambleside, Lake District
The use of Multi-Locus Sequence Typing (MLST) to study genetic variation
within Ralstonia solanacearum
J. Danial, G.S. Saddler & P. van de Graaf
2. The 4th International Bacterial Wilt Symposium, 2006, York, UK
Studies on the genetic diversity of the potato brown rot pathogen Ralstonia
solanacearum race 3/biovar 2A
J. Danial, R. C. McHugh & G. S. Saddler
3. The 8th Conference of the European Foundation for Plant Pathology
& BSPP Presidential Meeting, 2006, Copenhagen
Application of multi-locus sequence typing for characterisation of the
brown rot pathogen Ralstonia solanacearum
J. Danial, R. C. McHugh & G. S. Saddler
Posters
1. Society for General Microbiology, 2005, Heriot-Watt University, Edinburgh
The use of Multi-Locus Sequence Typing (MLST) to study genetic variation
within Ralstonia solanacearum
J. Danial, G.S. Saddler, P. van de Graaf & W.J. Mitchell
xi
2. The 11th International Conference on Plant Pathogenic Bacteria, 2006,
Edinburgh
Studies on the genetic diversity of the potato brown rot pathogen Ralstonia
solanacearum race 3/biovar 2A
J. Danial, R. C. McHugh & G. S. Saddler
3. The 7th Conference of the European Foundation for Plant Pathology & BSPP
Presidential Meeting, 2004, Aberdeen
The use of multi-locus sequence typing (MLST) to study genetic variation
within Ralstonia solanacearum
J. Danial, G.S. Saddler & P. van de Graaf
xii