FAAS Agricultural microbiology research team 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

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

FJAT FAAS Agricultural microbiology research team FJAT 1 、 Bacterial wilt disease and its pathogen forms Many researches has been made in the population dynamic, subspecies variation, molecular biology, gene regulation, etc. 。 Ralstonia solanacearum was found in 1864 ， it could infected many plants with about 44 family ， more than 300 species ， so called “plant cancer”. But the disease control would remain difficulties. The new idea was put forward to develop plant vaccin. (1) About bacterial wilt disease

FJAT FAAS Agricultural microbiology research team FJAT 1 、 Bacterial wilt disease and its pathogen forms RS Differentiation to (2) RS existed the virulent and avirulent strains, identified by TTC culture medium and plant infection. Virulent St. Virulent St. Avirulent St. Avirulent St. Infection and dead Infection and alive

FJAT FAAS Agricultural microbiology research team FJAT 1 、 Bacterial wilt disease and its pathogen forms (3) Pathogenicity index [PI] of virulent and avirulent RS in TTC medium in TTC medium 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. virulent interim avirulent Pathogenicity index Mortality (%)

FJAT FAAS Agricultural microbiology research team FJAT 1 、 Bacterial wilt disease and its pathogen forms (4) Pathogenicity divergency of RS populations infected in tissu culture plantlet of tomato G1: High infected mortaliy, short latent period, small PI <0.41 G2: Middle to high infected mortaliy, long latent period, middle PI= G3: No infected mortaliy, long latent period, large PI>0.80 。 。 。 。 。 。 。 。

FJAT FAAS Agricultural microbiology research team FJAT 2 、 Polymorphism of Ralstonia solanacearum (RS) (1) RAMS primers polymorphism analysis of Ralstonia solanacearum RAMS primers polymorphism Pu tian place Ning De place tomato egg plant pepper 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.

FJAT FAAS Agricultural microbiology research team FJAT 2 、 Polymorphism of Ralstonia solanacearum (RS) (2) BOX-PCR geographic polymorphism analysis of R. solanacearum λ=20 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. Ningde dist. Nanping dist. Fuzhou dist. Group 1 Group 2 Group 3 Tomato plants

FJAT FAAS Agricultural microbiology research team FJAT 2 、 Polymorphism of Ralstonia solanacearum (RS) (3) REP-PCR host polymorphism analysis of R. solanacearum λ=8 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. Egg plan+tomato Group 1 Chilli Group 2 +tomato Chilli+tomato Group 3 Ningde district

FJAT FAAS Agricultural microbiology research team FJAT 2 、 Polymorphism of Ralstonia solanacearum (RS) (4) PLFAs polymorphism analysis of R. solanacearum PLFAs of the virulent and avirulent RS Aviru lent Virul ent 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.

FJAT FAAS Agricultural microbiology research team FJAT 2 、 Polymorphism of Ralstonia solanacearum (RS) 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 , 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. Y 1 = X X X 13 Y 2 = X X X 13 Y 3 = X X X 13 (5) Discrimination analysis of PLFAs to distinguish the pathogenicity RS G1 ： avirulent G2 ： interim G3 ： virulent X 1 =C14:0, X 9 =C17:0, X 13 =C18:0 Cluster of the RS strains based on PLFAs

FJAT FAAS Agricultural microbiology research team FJAT 2 、 Polymorphism of Ralstonia solanacearum (RS) (6) Genome analysis of the virulent and avirulent RS strains

FJAT FAAS Agricultural microbiology research team FJAT 3 、 Characteristics of avirent Ralstonia solanacearum (1) Reproductive rate of the virulent and avirulent RS Cultural time Concentration (CFU/ml) Virulent strain Avirulent strain Before 24 hAfter 24 h Reproductive rate of the avirulent strain goes faster than the virulent before 24 h.

FJAT FAAS Agricultural microbiology research team FJAT 3 、 Characteristics of avirent Ralstonia solanacearum Control medium virulent avirulent EPS ug/ml virulent avirulent (2) Differences of EPS and protain between virulent and avirulent RS

FJAT FAAS Agricultural microbiology research team FJAT 3 、 Characteristics of avirent Ralstonia solanacearum (3) Differences of HPLC chromatogram between virulent and avirulent RS between virulent and avirulent RS

FJAT FAAS Agricultural microbiology research team FJAT 3 、 Characteristics of avirent Ralstonia solanacearum UV spectrophotometry (4) Differences of UV spectrophotometry between virulent and avirulent RS between virulent and avirulent RS

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

FJAT FAAS Agricultural microbiology research team FJAT 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 CUL: Cultural time can’t change RS to avirulent Subculture can’t change RS to avirulent BIO: Bacillus can change RS to avirulent

FJAT FAAS Agricultural microbiology research team FJAT 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 FAAS Agricultural microbiology research team FJAT 3 、 Characteristics of avirent Ralstonia solanacearum (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

FJAT FAAS Agricultural microbiology research team FJAT 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’ phcA4 ： 5’-GGAAGG CGTTGAGGTTGT-3’ Primer 2: phcA5 ： 5’-TGCCTTTCCACCTTCTGT-3’ phcA6 ： 5’-AGGCGTTGAGGTTGTAGGC-3’ phcA6 ： 5’-AGGCGTTGAGGTTGTAGGC-3’ Avirulent interim virulent non-RS 1-avirulent RS 2-interim toxicity RS 3-virulent RS 4-non RS

FJAT FAAS Agricultural microbiology research team FJAT 4 、 Construction and mechanism of avrient RS (1) Tn5 insertion to R. solanacearum Primer ： P1: 5′-GGTGCGACAATCTATCGA-3′ P2: 5′-CTCATCGAGCATCAAATG-3′ Rs91

FJAT FAAS Agricultural microbiology research team FJAT 4 、 Construction of of avirulent mutants by Tn5 transposon EZ-Tn5 Transposomes EZ-Tn5 Transposomes (1) Insertion of Tn5 transposon into R. solanacearum T2 T8 T7 T5 T4

FJAT FAAS Agricultural microbiology research team FJAT 4 、 Construction and mechanism of avrient RS Add Your Text concept Concept Concept Concept

FJAT FAAS Agricultural microbiology research team FJAT

FAAS Agricultural microbiology research team FJAT

FAAS Agricultural microbiology research team FJAT

FAAS Agricultural microbiology research team FJAT

FAAS Agricultural microbiology research team FJAT

FAAS Agricultural microbiology research team FJAT

FAAS Agricultural microbiology research team FJAT

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