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Polymorphism of Ralstonia
solanacearum and it’s plant vaccin
development
Prof. Dr. Liu Bo
Agricultural Microbiology Research Team
Bioresources research institute
Fujian Academy of Agricultural Sciences
fzliubo@163.com
25-06-2011
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Contents
1
Bacterial wilt disease and its pathogen forms
2
Polymorphism of Ralstonia solanacearum (RS)
3
Characteristics of avirent RS
4
Construction and mechanism of avrient RS
5
New idea of plant vaccin development
25-06-2011
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1、Bacterial wilt disease and its pathogen forms
(1) About bacterial wilt disease
Ralstonia solanacearum was found in 1864 ，
it could infected many plants with about 44
family，more than 300 species，so called
“plant cancer”.
Many researches has been made in the
population dynamic, subspecies variation,
molecular biology, gene regulation, etc.。
But the disease control would remain
difficulties . The new idea was put
forward to develop plant vaccin.
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1、Bacterial wilt disease and its pathogen forms
(2) RS existed the virulent and avirulent strains, identified by
TTC culture medium and plant infection.
RS
Differentiation to
Virulent St.
Avirulent St.
Infection and dead
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Infection and alive
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1、Bacterial wilt disease and its pathogen
forms
(%)
Mortality
发病率（%）
(3) Pathogenicity index [PI] of virulent and avirulent RS
in TTC medium
virulent
100
90
80
70
60
50
40
30
20
10
0
0
0.25
interim
0.5
avirulent
0.75
1
弱化指数
Pathogenicity index
Pathogenicity index was made up
by red diameter / whole colony
diameter
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RS strains were collected from
fields with the relationship
between pathogenicity index and
mortality.
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1、Bacterial wilt disease and its pathogen forms
(4) Pathogenicity divergency of RS populations infected in
tissu culture plantlet of tomato
G1: High infected
mortaliy, short
latent period, small
PI <0.41
。
。
。
。
。
。
。
。
。
。
。
。
G2: Middle to high
infected mortaliy,
long latent period,
middle PI=0.520.68
G3: No infected
mortaliy, long
latent period,
large PI>0.80
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2、Polymorphism of Ralstonia solanacearum (RS)
(1) RAMS primers polymorphism analysis of Ralstonia solanacearum
RAMS primers polymorphism
Pu
tian
place
tomato
egg plant
Cluster analysis based on
the RAMS bands showed
that 22 isolates could be
divided into 3 group with
characters of host crop
and climate zones.
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Ning
De
place
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pepper
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2、Polymorphism of Ralstonia solanacearum (RS)
(2) BOX-PCR geographic polymorphism analysis of R. solanacearum
Tomato plants
Group 1
Ningde dist.
The cluster analysis
showed that the BOXPCR genetic diversity of
R. solanacearum was
concerned on geographic origins with group
1 for Ningde district,
group 2 for Nanping district, group 3 for Fuzhou
district.
Group 2
Nanping dist.
Group 3
Fuzhou dist.
λ=20
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2、Polymorphism of Ralstonia solanacearum (RS)
(3) REP-PCR host polymorphism analysis of R. solanacearum
Ningde district
Group 1
Egg plan+tomato
The cluster analysis showed
that the REP-PCR genetic
diversity of R. solanacearum
was concerned on host with
group 1 for Egg plan+tomato
, group 2 for Pepper, group 3
for Pepper+tomato.
Group 2
Chilli
Group 3
Chilli+tomato
λ=8
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2、Polymorphism of Ralstonia solanacearum (RS)
(4) PLFAs polymorphism analysis of R. solanacearum
PLFAs of the virulent and avirulent RS
Aviru
lent
Virul
ent
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The PLFAs of A,
B, C, D, E, F, between the avirulent and virulent
RS to show the
avirulent with 0%
M., virulent with
30% M. and high
toxicity with
100% M.
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2、Polymorphism of Ralstonia solanacearum (RS)
(5) Discrimination analysis of PLFAs to distinguish the pathogenicity RS
G1：
avirulent
Y1=-16.3353 + 0.0012X1 + 0.0056X9 - 0.0016X13
Y2= -3.4928 + 0.0002X1 + 0.0003X9 + 0.0025X13
Y3= -9.1550 - 0.0003X1 + 0.0039X9 + 0.0042X13
X1=C14:0, X9=C17:0, X13=C18:0
G2：
interim
G3：
virulent
group 1 - avirulent strains with PI>0.80，
reinfected mortality 0% in the tomato
tissue culture plantlets .
group 2 - interim strains with PI in the range of
0.60-0.80, reinfected mortality
6.7%~ 100% in the tomato tissue
culture plantlets。
group 3 – virulent strains with PI<0.60,
reinfected mortality 100% in the
tomato tissue culture plantlets.
Cluster of the RS strains based on PLFAs
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2、Polymorphism of Ralstonia solanacearum (RS)
(6) Genome analysis of the virulent and avirulent RS strains
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3、Characteristics of avirent Ralstonia solanacearum
(1) Reproductive rate of the virulent and avirulent RS
孢子浓度（lgy)
(CFU/ml)
Concentration
10.00
8.00
强菌株
Virulent strain
弱菌株
Avirulent strain
6.00
4.00
2.00
Before 24 h
0.00
0
12
After 24 h
24
36
培养时间（h)
Cultural time
48
60
Reproductive rate of the avirulent strain goes faster than the
virulent before 24 h.
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3、Characteristics of avirent Ralstonia solanacearum
EPS ug/ml
(2) Differences of EPS and protain between virulent and avirulent RS
virulent
Control
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medium
virulent
avirulent
avirulent
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3、Characteristics of avirent Ralstonia solanacearum
(3) Differences of HPLC chromatogram
between virulent and avirulent RS
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3、Characteristics of avirent Ralstonia solanacearum
(4) Differences of UV spectrophotometry
between virulent and avirulent RS
UV spectrophotometry
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3、Characteristics of avirent Ralstonia solanacearum
(5) Differences of mortality between virulent and avirulent RS
100% M. in 7day
0% M. in 20day
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3、Characteristics of avirent Ralstonia solanacearum
(6) Factors effecting on the change from virulent to avirulent RS
CHEM: Antibiotic can’t
change RS to avirulent
PHYS: Ultrasonic can’t change
RS to avirulent
CUL: Cultural time can’t change RS to avirulent
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BIO: Bacillus can change
RS to avirulent
Subculture can’t change RS to avirulent
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3、Characteristics of avirent Ralstonia solanacearum
(7) Inhibit machenism of Bacillus cereus to v-RS and a-RS
Fermentation liquid (X4) of Bacillus cereus caused 99% mortality to virulent strain, 37%
mortality to avirulent strain. It meant that the virulent strain was more susceptible to
Bacillus cereus than the avirulent did.
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3、Characteristics of avirent Ralstonia solanacearum
(8) Simulation of inhibit effect of Bacillus cereus to v-RS and a-RS
1
3
4
2
Pure v-RS(1) and a-RS(2) mixed with B. cereus respectively resulted in (3) with two
strains growing together and in (4) with only a-RS gowing. It meant that B. cereus
could select the RS avirulent strain and suppress the RS virulent strain.
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3、Characteristics of avirent Ralstonia solanacearum
(9) Probe of phcA gene designed for distinguish v-RS and a-RS
Primer 1: phcA3：5’-CGACAACGAGTGGGGCTACTCGAAC-3’
phcA4：5’-GGAAGG CGTTGAGGTTGT-3’
Primer 2: phcA5：5’-TGCCTTTCCACCTTCTGT-3’
phcA6：5’-AGGCGTTGAGGTTGTAGGC-3’
1
3
Avirulent
virulent
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1
2
1
4
3
1
1-avirulent RS
2-interim toxicity RS
3-virulent RS
4-non RS
2
4
interim
non-RS
3
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1
4
4
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4、Construction and mechanism of avrient RS
(1) Tn5 insertion to R. solanacearum
Primer： P1: 5′-GGTGCGACAATCTATCGA-3′
P2: 5′-CTCATCGAGCATCAAATG-3′
Rs91
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4、Construction of of avirulent mutants by Tn5 transposon
(1) Insertion of Tn5 transposon into R. solanacearum
EZ-Tn5
Transposomes
T2
T4
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T8
T7
T5
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4、Construction and mechanism of avrient RS
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