Polymorphism of ralstonia solanacearum and it s plant vaccin development
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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 [email protected] 1. 2. 3. 4. 5. Bacterial wilt disease and its pathogen forms.

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Polymorphism of Ralstonia solanacearum and it’s plant vaccin development

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Polymorphism of ralstonia solanacearum and it s plant vaccin development

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]

Agricultural microbiology research team


Contents

1

2

3

4

5

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 avirulent

Contents

FJAT


1 bacterial wilt disease and its pathogen forms

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.

FJAT


1 bacterial wilt disease and its pathogen forms1

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 Infection and alive

FJAT


1 bacterial wilt disease and its pathogen forms2

1、Bacterial wilt disease and its pathogen forms

(3) Pathogenicity index [PI] of virulent and avirulent RS

in TTC medium

virulent interim avirulent

Mortality (%)

Pathogenicity index

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.

FJAT


1 bacterial wilt disease and its pathogen forms3

G1: High infected mortaliy, short latent period, small PI <0.41

G2: Middle to high infected mortaliy, long latent period,

middle PI=0.52-0.68

。 。 。。

。 。 。 。

。 。 。 。

G3: No infected mortaliy, long latent period, large PI>0.80

1、Bacterial wilt disease and its pathogen forms

(4) Pathogenicity divergency of RS populations infected in tissu culture plantlet of tomato

FJAT


2 polymorphism of ralstonia solanacearum rs

2、Polymorphism of Ralstonia solanacearum (RS)

(1) RAMS primers polymorphism analysis of Ralstonia solanacearum

RAMS primers polymorphism

tomato

Pu tian place

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.

Ning De place

pepper

FJAT


2 polymorphism of ralstonia solanacearum rs1

λ=20

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 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.

Group 2

Nanping dist.

Group 3

Fuzhou dist.

FJAT


2 polymorphism of ralstonia solanacearum rs2

λ=8

2、Polymorphism of Ralstonia solanacearum (RS)

(3) REP-PCR hostpolymorphism 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

FJAT


2 polymorphism of ralstonia solanacearum rs3

2、Polymorphism of Ralstonia solanacearum (RS)

(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.

Avirulent

Virulent

FJAT


2 polymorphism of ralstonia solanacearum rs4

2、Polymorphism of Ralstonia solanacearum (RS)

(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

G1:avirulent

X1=C14:0, X9=C17:0, X13=C18:0

G2:interim

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.

G3:virulent

Cluster of the RS strains based on PLFAs

FJAT


2 polymorphism of ralstonia solanacearum rs5

2、Polymorphism of Ralstonia solanacearum (RS)

(6) Genome analysis of the virulent and avirulent RS strains

FJAT


3 characteristics of avirent ralstonia solanacearum

3、Characteristics of avirent Ralstonia solanacearum

(1) Reproductive rate of the virulent and avirulent RS

Virulent strain

Avirulent strain

Concentration (CFU/ml)

Before 24 h

After 24 h

Cultural time

Reproductive rate of the avirulent strain goes faster than the virulent before 24 h.

FJAT


3 characteristics of avirent ralstonia solanacearum1

3、Characteristics of avirent Ralstonia solanacearum

(2) Differences of EPS and protain between virulent and avirulent RS

virulent avirulent

EPS ug/ml

Control medium virulent avirulent

FJAT


3 characteristics of avirent ralstonia solanacearum2

3、Characteristics of avirent Ralstonia solanacearum

(3) Differences of HPLC chromatogram

between virulent and avirulent RS

FJAT


3 characteristics of avirent ralstonia solanacearum3

3、Characteristics of avirent Ralstonia solanacearum

(4) Differences of UV spectrophotometry

between virulent and avirulent RS

UV spectrophotometry

FJAT


3 characteristics of avirent ralstonia solanacearum4

3、Characteristics of avirent Ralstonia solanacearum

(5) Differences of mortality between virulent and avirulent RS

100% M. in 7day

0% M. in 20day

FJAT


3 characteristics of avirent ralstonia solanacearum5

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

BIO: Bacillus can change RS to avirulent

Subculture can’t change RS to avirulent

CUL: Cultural time can’t change RS to avirulent

FJAT


3 characteristics of avirent ralstonia solanacearum6

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.

FJAT


3 characteristics of avirent ralstonia solanacearum7

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.

FJAT


3 characteristics of avirent ralstonia solanacearum8

1

1

4

4

3

3

4

1

2

1

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-avirulent RS

2-interim toxicity RS

3-virulent RS

4-non RS

1 2 3 4

Avirulent interim virulent non-RS

FJAT


4 construction and mechanism of avrient rs

Rs91

4、Construction and mechanism of avrient RS

(1) Insertion of Tn5 transposon toR. solanacearum

Kanamycin resistant gene

Primer: P1: 5′-GGTGCGACAATCTATCGA-3′

P2: 5′-CTCATCGAGCATCAAATG-3′

1004 mutants

EZ-Tn5™ <KAN-2> Insertion Kit EPICENTR

FJAT


4 construction of of avirulent mutants by tn5 transposon

4、Construction of of avirulent mutants by Tn5 transposon

(2) Tn5 transposon mutagenesis of avirulent R. solanacearum

EZ-Tn5 Transposomes

T8

T2

T7

T4

T5

FJAT


4 construction of of avirulent mutants by tn5 transposon1

4、Construction of of avirulent mutants by Tn5 transposon

(3) Amplification of flanking secquences of avirulent mutants

T4

T8

T2

T5

T7

Amplification of flanking secquences of avirulent mutants T2(a-1)、T4(b-2)、T5(b-1)、T7(a-2)和T8(c-1).

FJAT


4 construction of of avirulent mutants by tn5 transposon2

4、Construction of of avirulent mutants by Tn5 transposon

(4) The insertion of avirulent mutants T2、T4、T5、T7 and T8

RED PARTS: ME =Mosaic End from Tn5 transposon 'AGATGTGTATAAGAGACAG 3'

FJAT


4 construction of of avirulent mutants by tn5 transposon3

4、Construction of of avirulent mutants by Tn5 transposon

(5) The insertion position of avirulent mutants T2、T5、T7 and T8

T2

1048

1097

1489

T5

T7

T8

FJAT


4 construction of of avirulent mutants by tn5 transposon4

4、Construction of of avirulent mutants by Tn5 transposon

(6) Growth curve of avirulent mutants T2、T4、T5、T7 and T8

FJAT


4 construction of of avirulent mutants by tn5 transposon5

4、Construction of of avirulent mutants by Tn5 transposon

(7) Effect of pH on avirulent mutants T2、T4、T5、T7 and T8

FJAT


4 construction of of avirulent mutants by tn5 transposon6

4、Construction of of avirulent mutants by Tn5 transposon

(8) The relative content of EPSI of avirulent mutants

FJAT


4 construction of of avirulent mutants by tn5 transposon7

4、Construction of of avirulent mutants by Tn5 transposon

(9) Pathogencity of avirulent mutents infected in tomato in the 15th day

FJAT


5 new idea of plant vaccin development with avirulent

A-RS

5、New idea of plant vaccin development with avirulent

(1) Idea of plant vaccin development with avrirulent RS

Tn5 insertion

GPS marked

Infect mechanism

Field experiment

Plant vaccin production

Effiency

Innoculation

FJAT


Polymorphism of ralstonia solanacearum and it s plant vaccin development

(2) GFP transformation of Tn5-inserted avirulent RS

fluorescence microscope

plasmid

transformant

Morphology of

bacterial body

morphology of

colony

Molecular detection

FJAT


Polymorphism of ralstonia solanacearum and it s plant vaccin development

(3) Infect mechanism of GFP-marked avirulent RS

leaf

stem

root

24 h 48 h 72 h

FJAT


Polymorphism of ralstonia solanacearum and it s plant vaccin development

(4) Enzyme activity of tomato seedling Infected with avirulent RS

25 days

25 days

25 days

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

FJAT


Polymorphism of ralstonia solanacearum and it s plant vaccin development

(5) Products of plant vaccin produced with an avirulent RS

FJAT


Polymorphism of ralstonia solanacearum and it s plant vaccin development

(6) Protect effiency of bacterial wilt disease by

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%

FJAT


Polymorphism of ralstonia solanacearum and it s plant vaccin development

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Polymorphism of ralstonia solanacearum and it s plant vaccin development

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Polymorphism of ralstonia solanacearum and it s plant vaccin development

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Polymorphism of ralstonia solanacearum and it s plant vaccin development

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Polymorphism of ralstonia solanacearum and it s plant vaccin development

FJAT


Polymorphism of ralstonia solanacearum and it s plant vaccin development

FJAT


Polymorphism of ralstonia solanacearum and it s plant vaccin development

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Polymorphism of ralstonia solanacearum and it s plant vaccin development

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Polymorphism of ralstonia solanacearum and it s plant vaccin development

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