FAAS 25-06-2011 Agricultural microbiology research team Polymorphism of Ralstonia solanacearum and it’s plant vaccin development Prof. Dr. Liu Bo Agricultural

25-06-2011 Agricultural microbiology research team

FAAS

Polymorphism of Polymorphism of Ralstonia Ralstonia solanacearumsolanacearum and it’s plant vaccin and it’s plant vaccin

development development

Prof. Dr. Liu BoProf. Dr. Liu Bo

Agricultural Microbiology Research TeamAgricultural Microbiology Research Team

Bioresources research instituteBioresources research institute

Fujian Academy of Agricultural SciencesFujian Academy of Agricultural Sciences

[email protected]@163.com

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Agricultural microbiology research team

ContentsContents

Bacterial wilt disease and its pathogen forms Bacterial wilt disease and its pathogen forms 11

Polymorphism of Polymorphism of Ralstonia solanacearum Ralstonia solanacearum (RS) (RS) 22

Characteristics of avirulent Characteristics of avirulent Ralstonia solanacearumRalstonia solanacearum 33

Construction of avirulent Construction of avirulent Ralstonia solanacearumRalstonia solanacearum 44

New idea of plant vaccin development with avirulent New idea of plant vaccin development with avirulent

55

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11、、 Bacterial wilt disease and its pathogen formsBacterial wilt disease and its pathogen forms

Many researches has been made in the Many researches has been made in the population dynamic, subspecies variation, population dynamic, subspecies variation, molecular biology, gene regulation, etc.molecular biology, gene regulation, etc. 。。

Ralstonia solanacearumRalstonia solanacearum was found in 1864 was found in 1864 ，，it could infected many plants with about 44 it could infected many plants with about 44 familyfamily，， more than 300 speciesmore than 300 species，， so so called called ““plant cancer”.plant cancer”.

But the disease control would remain But the disease control would remain difficulties . The new idea was put difficulties . The new idea was put forward to develop plant vaccin.forward to develop plant vaccin.

(1) About bacterial wilt disease (1) About bacterial wilt disease

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11、、 Bacterial wilt disease and its pathogen formsBacterial wilt disease and its pathogen forms

RSRSDifferentiation toDifferentiation to

(2) RS existed the virulent and avirulent strains, identified by (2) RS existed the virulent and avirulent strains, identified by TTC culture medium and plant infection.TTC culture medium and plant infection.

Virulent St. Virulent St.

Avirulent St. Avirulent St.

Infection and dead Infection and dead Infection and aliveInfection and alive

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11、、 Bacterial wilt disease and its pathogen Bacterial wilt disease and its pathogen formsforms

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

0102030405060708090

100

0 0. 25 0. 5 0. 75 1

弱化指数

%发病率（

）Pathogenicity index was made up Pathogenicity index was made up by red diameter / whole colony by red diameter / whole colony diameterdiameter

RS strains were collected from RS strains were collected from fields with the relationship fields with the relationship between pathogenicity index and between pathogenicity index and mortality. mortality.

virulent interim avirulentvirulent interim avirulent

Pathogenicity index Pathogenicity index

Mor

talit

y (%

) M

orta

lity

(%)

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11、、 Bacterial wilt disease and its pathogen formsBacterial wilt disease and its pathogen forms(4) Pathogenicity divergency of RS populations infected in (4) Pathogenicity divergency of RS populations infected in

tissu culture plantlet of tomatotissu culture plantlet of tomato

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

G2: Middle to high G2: Middle to high infected mortaliy, infected mortaliy, long latent period,long latent period,middle PI=0.52-middle PI=0.52-0.68 0.68

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

。。。。。。。。。。。。。。。。。。。。。。。。

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22、、 Polymorphism of Polymorphism of Ralstonia solanacearum Ralstonia solanacearum (RS)(RS)

(1) RAMS primers polymorphism analysis of (1) RAMS primers polymorphism analysis of Ralstonia solanacearumRalstonia solanacearum

RAMS primers polymorphism

Pu tian place

Ning De place

tomatotomato

egg plantegg plant

pepperpepper

Cluster analysis based on Cluster analysis based on the RAMS bands showed the RAMS bands showed that 22 isolates could be that 22 isolates could be divided into 3 group with divided into 3 group with characters of host crop characters of host crop and climate zones. and climate zones.

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(2) BOX-PCR geographic polymorphism analysis of (2) BOX-PCR geographic polymorphism analysis of R. solanacearumR. solanacearum

λ=20

The cluster analysis The cluster analysis showed that the BOX-showed that the BOX-PCR genetic diversity of PCR genetic diversity of R. solanacearum R. solanacearum was was concerned on geogra- concerned on geographic origins with group phic origins with group 1 for Ningde district, 1 for Ningde district, group 2 for Nanping dis-group 2 for Nanping dis-trict, group 3 for Fuzhou trict, group 3 for Fuzhou district. district.

Ningde dist.Ningde dist.

Nanping dist.Nanping dist.

Fuzhou dist.Fuzhou dist.

Group 1Group 1

Group 2Group 2

Group 3Group 3

Tomato plants Tomato plants

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(3) REP-PCR host(3) REP-PCR host polymorphism analysis of polymorphism analysis of R. solanacearumR. solanacearum

λ=8

The cluster analysis showed The cluster analysis showed that the REP-PCR genetic that the REP-PCR genetic diversity of diversity of R. solanacearum R. solanacearum was concerned on host with was concerned on host with group 1 for Egg plan+tomatogroup 1 for Egg plan+tomato, group 2 for Pepper, group 3 , group 2 for Pepper, group 3 for Pepper+tomato. for Pepper+tomato.

Egg plan+tomatoEgg plan+tomatoGroup 1Group 1

ChilliGroup 2Group 2

Chilli+tomato+tomatoGroup 3Group 3

Ningde districtNingde district

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(4) PLFAs(4) PLFAs polymorphism analysis of polymorphism analysis of R. solanacearumR. solanacearum

PLFAs of the virulent and avirulent RS PLFAs of the virulent and avirulent RS

AviruAvirulent lent

VirulVirulent ent

The PLFAs of A, The PLFAs of A,

B, C, D, E, F, bet-B, C, D, E, F, bet-

ween the aviru-ween the aviru-

lent and virulent lent and virulent

RS to show the RS to show the

avirulent with 0% avirulent with 0%

M., virulent with M., virulent with

30% M. and high 30% M. and high

toxicity with toxicity with

100% M. 100% M.

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group 1 - avirulent strains with group 1 - avirulent strains with PI>0.80PI>0.80，， reinfected mortality 0% in reinfected mortality 0% in the tomato tissue culture plantlets .the tomato tissue culture plantlets .

group 2 - interim strains with PI in the range of group 2 - interim strains with PI in the range of 0.60-0.80, 0.60-0.80, reinfected mortality reinfected mortality 6.7%~ 100%6.7%~ 100% in the tomato tissue in the tomato tissue culture plantletsculture plantlets 。。

group 3 – virulent strains with PI<0.60, group 3 – virulent strains with PI<0.60, reinfected mortality 100% in the reinfected mortality 100% in the tomato tissue culture plantlets.tomato tissue culture plantlets.

YY11=-16.3353 + 0.0012X=-16.3353 + 0.0012X1 1 + 0.0056X+ 0.0056X9 9 - -

0.0016X0.0016X1313

YY22= -3.4928 + 0.0002X= -3.4928 + 0.0002X1 1 + 0.0003X+ 0.0003X9 9 + +

0.0025X0.0025X1313

YY33= -9.1550 - 0.0003X= -9.1550 - 0.0003X1 1 + 0.0039X+ 0.0039X9 9 + +

0.0042X0.0042X1313

(5) Discrimination(5) Discrimination analysis of PLFAs to distinguish the pathogenicity RSanalysis of PLFAs to distinguish the pathogenicity RS

G1G1：：avirulentavirulent

G2G2：：interiminterim

G3G3：：virulentvirulent

XX11=C14:0, X=C14:0, X99=C17:0, X=C17:0, X1313=C18:0=C18:0

Cluster of the RS strains based on PLFAs Cluster of the RS strains based on PLFAs

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

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33、、 Characteristics of avirent Characteristics of avirent Ralstonia solanacearum Ralstonia solanacearum

0. 00

2. 00

4. 00

6. 00

8. 00

10. 00

0 12 24 36 48 60

h)培养时间（

lgy)

孢子浓度（

强菌株弱菌株

(1) Reproductive rate of the virulent and avirulent RS (1) Reproductive rate of the virulent and avirulent RS

Cultural time Cultural time

Con

cent

ratio

n (C

FU/m

l)C

once

ntra

tion

(CFU

/ml)

Virulent strain Virulent strain

Avirulent strainAvirulent strain

Before 24 h After 24 h

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

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33、、 Characteristics of avirent Characteristics of avirent Ralstonia solanacearumRalstonia solanacearum

Control medium virulent avirulentControl medium virulent avirulent

EPS

ug/m

lEP

S ug

/ml

virulent avirulent virulent avirulent

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

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(3) Differences of HPLC chromatogram (3) Differences of HPLC chromatogram between virulent and avirulent RS between virulent and avirulent RS

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UV spectrophotometryUV spectrophotometry

(4) Differences of UV spectrophotometry (4) Differences of UV spectrophotometry between virulent and avirulent RS between virulent and avirulent RS

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(5) Differences of mortality between virulent and avirulent RS (5) Differences of mortality between virulent and avirulent RS

100% M. in 7day100% M. in 7day

0% M. in 20day0% M. in 20day

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(6) Factors effecting on the change from virulent to avirulent RS (6) Factors effecting on the change from virulent to avirulent RS

CHEM: Antibiotic can’t CHEM: Antibiotic can’t change RS to avirulent change RS to avirulent

PHYS: Ultrasonic can’t change PHYS: Ultrasonic can’t change RS to avirulentRS to avirulent

CUL: Cultural time can’t change RS to avirulent CUL: Cultural time can’t change RS to avirulent SubcultureSubculture can’t change RS to avirulent can’t change RS to avirulent

BIO: Bacillus can change BIO: Bacillus can change RS to avirulent RS to avirulent

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(7) Inhibit machenism of (7) Inhibit machenism of Bacillus cereusBacillus cereus to v-RS and a-RS to v-RS and a-RS

Fermentation liquid (X4) of Fermentation liquid (X4) of Bacillus cereusBacillus cereus caused 99% mortality to virulent strain, 37% caused 99% mortality to virulent strain, 37% mortality to avirulent strain. It meant that the virulent strain was more susceptible to mortality to avirulent strain. It meant that the virulent strain was more susceptible to Bacillus cereusBacillus cereus than the avirulent did. than the avirulent did.

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(8) Simulation of inhibit effect of (8) Simulation of inhibit effect of Bacillus cereusBacillus cereus to v-RS and a-RS to v-RS and a-RS

Pure v-RS(1) and a-RS(2) mixed with B. cereus respectively resulted in (3) with two 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 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. could select the RS avirulent strain and suppress the RS virulent strain.

1

2

3 4

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(9) Probe of phcA gene designed for distinguish v-RS and a-RS (9) Probe of phcA gene designed for distinguish v-RS and a-RS PPrimer 1: phcA3rimer 1: phcA3：： 5’-CGACAACGAGTGGGGCTACTCGAAC-3’5’-CGACAACGAGTGGGGCTACTCGAAC-3’ phcA4phcA4：： 5’-GGAAGG CGTTGAGGTTGT-3’5’-GGAAGG CGTTGAGGTTGT-3’Primer 2:Primer 2: phcA5phcA5：： 5’-TGCCTTTCCACCTTCTGT-3’5’-TGCCTTTCCACCTTCTGT-3’ phcA6phcA6：： 5’-AGGCGTTGAGGTTGTAGGC-3’5’-AGGCGTTGAGGTTGTAGGC-3’

11 11 4444 1133 3322 11 44

1 2 3 1 2 3 4 4Avirulent interim virulent Avirulent interim virulent non-RS non-RS

1-avirulent RS1-avirulent RS2-interim toxicity RS2-interim toxicity RS3-virulent RS 3-virulent RS 4-non RS4-non RS

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44、、 Construction and mechanism of avrient Construction and mechanism of avrient RSRS

(1) Insertion of Tn5 transposon to (1) Insertion of Tn5 transposon to R. solanacearumR. solanacearum PrimerPrimer ：： P1: 5′-GGTGCGACAATCTATCGA-3′P1: 5′-GGTGCGACAATCTATCGA-3′ P2: 5′-CTCATCGAGCATCAAATG-3′P2: 5′-CTCATCGAGCATCAAATG-3′

Rs91Rs91

Kanamycin Kanamycin resistant generesistant gene

1004 1004 mutantsmutants

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

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44、、 Construction of of avirulent mutants by Tn5 Construction of of avirulent mutants by Tn5 transposon transposon

EZ-Tn5 EZ-Tn5 Transposomes Transposomes

(2) Tn5 transposon mutagenesis of avirulent (2) Tn5 transposon mutagenesis of avirulent R. solanacearumR. solanacearum

T2T2 T8T8

T7T7T5T5

T4T4

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44、、 Construction of of avirulent mutants by Tn5 transposonConstruction of of avirulent mutants by Tn5 transposon

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

(3) Amplification of flanking secquences of avirulent mutants(3) Amplification of flanking secquences of avirulent mutants

T2T2

T7T7

T4T4

T5T5

T8T8

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

(4) The insertion of avirulent mutants T2(4) The insertion of avirulent mutants T2、、 T4T4、、 T5T5、、 T7 and T8T7 and T8

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44、、 Construction of of avirulent mutants by Tn5 Construction of of avirulent mutants by Tn5 transposontransposon

(5) The insertion position of avirulent mutants T2(5) The insertion position of avirulent mutants T2 、、 T5T5、、 T7 and T8 T7 and T8

10481048

T2T2

1489148910971097

T5T5 T7T7 T8T8

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(6) Growth curve of avirulent mutants T2(6) Growth curve of avirulent mutants T2 、、 T4T4、、 T5T5、、 T7 and T8T7 and T8

0. 0

0. 5

1. 0

1. 5

2. 0

2. 5

0 5 10 15 20 25 30 35

(h)时间t i me(h)

A 600

T2 T4 T5 T7 T8 Rs91(CK)

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-1.0

0.0

1.0

2.0

3.0

5 6 7 8 9pH值

A600

T2 T4 T5 T7 T8 Rs91(CK)

(7) Effect of pH on avirulent mutants (7) Effect of pH on avirulent mutants T2T2、、 T4T4、、 T5T5、、 T7 and T8T7 and T8

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0

5

10

15

20

25

30

35

40

Rs91(CK) T2 T4 T5 T7 T8Strains

Rel

ative

con

tent

of

EPS

I（

μg/O

D6

00 ）

(8) The relative content of EPSI of avirulent mutants (8) The relative content of EPSI of avirulent mutants

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

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55、、 New idea of plant vaccin development with avirulentNew idea of plant vaccin development with avirulent

(1) Idea of plant vaccin development with avrirulent RS(1) Idea of plant vaccin development with avrirulent RS

Tn5 Tn5 insertioninsertion

Infect Infect mechanism mechanism

GPS GPS marked marked

Effiency Effiency Innoculation Innoculation

Plant vaccin Plant vaccin productionproduction

A-A-RSRS

Field Field experimentexperiment

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(2) GFP transformation of Tn5-inserted avirulent RS(2) GFP transformation of Tn5-inserted avirulent RS

plasmidplasmid transformanttransformant

morphology of morphology of colonycolony

Morphology of Morphology of bacterial bodybacterial body

Molecular detectionMolecular detection

fluorescence microscopefluorescence microscope

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(3) Infect mechanism of GFP-marked avirulent RS (3) Infect mechanism of GFP-marked avirulent RS

24 h 48 h 72 h24 h 48 h 72 h

leafleaf

rootroot

stemstem

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0

20

40

60

80

100

120

140

接种对照接种对照接种对照

SOD

U•g-

1•mi

n-1

FW酶总活性（

）

0

50

100

150

200

250

300

350


POD

U•g-

1•mi

n-1

FW酶总活性（

）

900

950

1000

1050

1100

1150

1200

1250


PPO酶总活性（

U•g

-1•m

in-1

FW）

Effects of Ralstonia solanacearum strain FJAT-1458 on total POD activity in different parts of tomato

T ck T ck T ckT ck T ck T ckT ck T ck T ckT ck T ck T ckT ck T ck T ckT 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 PPO activity in different parts of tomato

(4) (4) EEnzyme activity of tomato seedling Infected with avirulent RS nzyme activity of tomato seedling Infected with avirulent RS

25 days 25 days 25 days 25 days 25 days 25 days

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

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(6) Protect effiency of bacterial wilt disease by (6) Protect effiency of bacterial wilt disease by inoculated with an avirulent RS in semi experiments inoculated with an avirulent RS in semi experiments PPlant vaccin AVIRULENT lant vaccin AVIRULENT was inoculated into was inoculated into tomato seedling in 10tomato seedling in 1044 cfu/mL, with water as cfu/mL, with water as control. 3 days after control. 3 days after treatment the seedling treatment the seedling was infected with virulent was infected with virulent RS, the results showed RS, the results showed that the infected plant that the infected plant vaccin treatment makes 0 vaccin treatment makes 0 dead, while that without dead, while that without plant vaccin treatment plant vaccin treatment caused 100% dead. caused 100% dead.

PPlant vaccin Water control Plant vaccinlant vaccin Water control Plant vaccin

0% 100% 0%0% 100% 0%

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25-06-2011 Agricultural microbiology research team

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FAAS 25-06-2011 Agricultural microbiology research team Polymorphism of Ralstonia solanacearum and it’s plant vaccin development Prof. Dr. Liu Bo Agricultural