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 [email protected] 1. 2. 3. 4. 5. Bacterial wilt disease and its pathogen forms.
Bacterial wilt disease and its pathogen forms
Polymorphism of Ralstonia solanacearum (RS)
Characteristics of avirulent Ralstonia solanacearum
Construction of avirulent Ralstonia solanacearum
New idea of plant vaccin development with avirulentContents
(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.
(2) RS existed the virulent and avirulent strains, identified by TTC culture medium and plant infection.
Infection and dead Infection and alive
(3) Pathogenicity index [PI] of virulent and avirulent RS
in TTC medium
virulent interim avirulent
Pathogenicity index was made up by red diameter / whole colony diameter
RS strains were collected from fields with the relationship between pathogenicity index and mortality.
G2: Middle to high infected mortaliy, long latent period,
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G3: No infected mortaliy, long latent period, large PI>0.801、Bacterial wilt disease and its pathogen forms
(4) Pathogenicity divergency of RS populations infected in tissu culture plantlet of tomato
(1) RAMS primers polymorphism analysis of Ralstonia solanacearum
RAMS primers polymorphism
Pu tian place
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.
Ning De place
λ=20 <0.412、Polymorphism of Ralstonia solanacearum (RS)
(2) BOX-PCR geographic polymorphism analysis of R. solanacearum
The cluster analysis showed that the BOX-PCR genetic diversity of R. solanacearum was concerned on geogra- phic origins with group 1 for Ningde district, group 2 for Nanping dis-trict, group 3 for Fuzhou district.
λ=8 <0.412、Polymorphism of Ralstonia solanacearum (RS)
(3) REP-PCR hostpolymorphism analysis of R. solanacearum
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.
(4) PLFAspolymorphism analysis of R. solanacearum
PLFAs of the virulent and avirulent RS
The PLFAs of A, B, C, D, E, F, bet-ween the aviru-lent and virulent RS to show the avirulent with 0% M., virulent with 30% M. and high toxicity with 100% M.
(5) Discriminationanalysis of PLFAs to distinguish the pathogenicity RS
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
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
(6) Genome analysis of the virulent and avirulent RS strains
(1) Reproductive rate of the virulent and avirulent RS
Before 24 h
After 24 h
Reproductive rate of the avirulent strain goes faster than the virulent before 24 h.
(2) Differences of EPS and protain between virulent and avirulent RS
Control medium virulent avirulent
(3) Differences of HPLC chromatogram
between virulent and avirulent RS
(4) Differences of UV spectrophotometry
between virulent and avirulent RS
(5) Differences of mortality between virulent and avirulent RS
100% M. in 7day
0% M. in 20day
(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
BIO: Bacillus can change RS to avirulent
Subculture can’t change RS to avirulent
CUL: Cultural time can’t change RS to avirulent
(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.
(8) Simulation of inhibit effect of Bacillus cereus to v-RS and a-RS
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.
13、Characteristics of avirent Ralstonia solanacearum
(9) Probe of phcA gene designed for distinguish v-RS and a-RS
Primer 1: phcA3：5’-CGACAACGAGTGGGGCTACTCGAAC-3’
2-interim toxicity RS
1 2 3 4
Avirulent interim virulent non-RS
Rs91 <0.414、Construction and mechanism of avrient RS
(1) Insertion of Tn5 transposon toR. solanacearum
Kanamycin resistant gene
Primer： P1: 5′-GGTGCGACAATCTATCGA-3′
EZ-Tn5™ <KAN-2> Insertion Kit EPICENTR
(2) Tn5 transposon mutagenesis of avirulent R. solanacearum
(3) Amplification of flanking secquences of avirulent mutants
Amplification of flanking secquences of avirulent mutants T2(a-1)、T4(b-2)、T5(b-1)、T7(a-2)和T8(c-1).
(4) The insertion of avirulent mutants T2、T4、T5、T7 and T8
RED PARTS: ME =Mosaic End from Tn5 transposon 'AGATGTGTATAAGAGACAG 3'
(5) The insertion position of avirulent mutants T2、T5、T7 and T8
(6) Growth curve of avirulent mutants T2、T4、T5、T7 and T8
(7) Effect of pH on avirulent mutants T2、T4、T5、T7 and T8
(8) The relative content of EPSI of avirulent mutants
(9) Pathogencity of avirulent mutents infected in tomato in the 15th day
A-RS <0.415、New idea of plant vaccin development with avirulent
(1) Idea of plant vaccin development with avrirulent RS
Plant vaccin production
24 h 48 h 72 h
(4) E <0.41 nzyme activity of tomato seedling Infected with avirulent RS
T ck T ck T ck
T ck T ck T ck
T ck T ck T ck
Effects of Ralstonia solanacearum strain FJAT-1458 on total SOD activity in different parts of tomato
Effects of Ralstonia solanacearum strain FJAT-1458 on total POD activity in different parts of tomato
Effects of Ralstonia solanacearum strain FJAT-1458 on total PPO activity in different parts of tomato
inoculated with an avirulent RS in semi experiments
Plant vaccin AVIRULENT was inoculated into tomato seedling in 104 cfu/mL, with water as control. 3 days after treatment the seedling was infected with virulent RS, the results showed that the infected plant vaccin treatment makes 0 dead, while that without plant vaccin treatment caused 100% dead.
Plant vaccin Water control Plant vaccin
0% 100% 0%
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Agricultural microbiology research team