Polymorphism of Ralstonia solanacearum and it’s plant vaccin development

1. 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 Agricultural microbiology research team

2. 1 2 3 4 5 Bacterial wilt disease and its pathogen forms Polymorphism of Ralstonia solanacearum (RS) Characteristics of avirent RS Construction and mechanism of avrient RS New idea of plant vaccin development Contents FJAT

3. 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

4. 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

5. 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

6. 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

7. 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

8. 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

9. 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

10. 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

11. 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

12. 2、Polymorphism of Ralstonia solanacearum (RS) (6) Genome analysis of the virulent and avirulent RS strains FJAT

13. 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

14. 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

15. 3、Characteristics of avirent Ralstonia solanacearum (3) Differences of HPLC chromatogram between virulent and avirulent RS FJAT

16. 3、Characteristics of avirent Ralstonia solanacearum (4) Differences of UV spectrophotometry between virulent and avirulent RS UV spectrophotometry FJAT

17. 3、Characteristics of avirent Ralstonia solanacearum (5) Differences of mortality between virulent and avirulent RS 100% M. in 7day 0% M. in 20day FJAT

18. 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

19. 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

20. 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

21. 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

22. Rs91 4、Construction and mechanism of avrient RS (1) Tn5 insertion toR. solanacearum Primer： P1: 5′-GGTGCGACAATCTATCGA-3′ P2: 5′-CTCATCGAGCATCAAATG-3′ FJAT

23. 4、Construction of of avirulent mutants by Tn5 transposon (1) Insertion of Tn5 transposon intoR. solanacearum EZ-Tn5 Transposomes T8 T2 T7 T4 T5 FJAT

24. Concept concept Concept Concept 4、Construction and mechanism of avrient RS Add Your Text Add Your Text FJAT

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