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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 fzliubo@163.com. 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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  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 avirulent Ralstonia solanacearum Construction of avirulent Ralstonia solanacearum New idea of plant vaccin development with avirulent 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) 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

  23. 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

  24. 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

  25. 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

  26. 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

  27. 4、Construction of of avirulent mutants by Tn5 transposon (6) Growth curve of avirulent mutants T2、T4、T5、T7 and T8 FJAT

  28. 4、Construction of of avirulent mutants by Tn5 transposon (7) Effect of pH on avirulent mutants T2、T4、T5、T7 and T8 FJAT

  29. 4、Construction of of avirulent mutants by Tn5 transposon (8) The relative content of EPSI of avirulent mutants FJAT

  30. 4、Construction of of avirulent mutants by Tn5 transposon (9) Pathogencity of avirulent mutents infected in tomato in the 15th day FJAT

  31. 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

  32. (2) GFP transformation of Tn5-inserted avirulent RS fluorescence microscope plasmid transformant Morphology of bacterial body morphology of colony Molecular detection FJAT

  33. (3) Infect mechanism of GFP-marked avirulent RS leaf stem root 24 h 48 h 72 h FJAT

  34. (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

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

  36. (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

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  46. www.themegallery.com Click to edit company slogan . Thank You ! Agricultural microbiology research team

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