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 avirulent Ralstonia solanacearum 3 construction of avirulent Ralstonia solanacearum 4 New idea of plant vaccin development with avirulent 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 tissue 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 ， infected mortality 0% in the tomato tissue culture plantlets. group 2 - interim strains with PI in the range of , infected mortality 6.7%~ 100% in the tomato tissue culture plantlets 。 group 3 – virulent strains with PI<0.60, infected 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 RS91 and avirulent RS98

FJAT FAAS Agricultural microbiology research team FJAT 3 、 Characteristics of avirulent 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 EPS difference between v- an a-RS virulent avirulent

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) Insertion of Tn5 transposon to R. solanacearum Primer ： P1: 5′-GGTGCGACAATCTATCGA-3′ P2: 5′-CTCATCGAGCATCAAATG-3′ P2: 5′-CTCATCGAGCATCAAATG-3′ Rs91 Kanamycin resistant gene 1004 mutants EZ-Tn5™ Insertion Kit EPICENTR

FJAT FAAS Agricultural microbiology research team FJAT 4 、 Construction of of avirulent mutants by Tn5 transposon EZ-Tn5 Transposomes EZ-Tn5 Transposomes (2) Tn5 transposon mutagenesis of avirulent R. solanacearum T2 T8 T7 T5 T4

FJAT FAAS Agricultural microbiology research team FJAT 4 、 Construction of of avirulent mutants by Tn5 transposon Amplification of flanking secquences of avirulent mutants T2(a-1) 、 T4(b-2) 、 T5(b-1) 、 T7(a-2) 和 T8(c-1). (3) Amplification of flanking secquences of avirulent mutants T2 T7 T4 T5 T8

FJAT FAAS Agricultural microbiology research team FJAT 4 、 Construction of of avirulent mutants by Tn5 transposon RED PARTS: ME =Mosaic End from Tn5 transposon 'AGATGTGTATAAGA GACAG 3' (4) The insertion of avirulent mutants T2 、 T4 、 T5 、 T7 and T8

FJAT FAAS Agricultural microbiology research team FJAT 4 、 Construction of of avirulent mutants by Tn5 transposon (5) The insertion position of avirulent mutants T2 、 T5 、 T7 and T T T5T7T8

FJAT FAAS Agricultural microbiology research team FJAT 4 、 Construction of of avirulent mutants by Tn5 transposon (6) Growth curve of avirulent mutants T2 、 T4 、 T5 、 T7 and T8

FJAT FAAS Agricultural microbiology research team FJAT 4 、 Construction of of avirulent mutants by Tn5 transposon (7) Effect of pH on avirulent mutants T2 、 T4 、 T5 、 T7 and T8

FJAT FAAS Agricultural microbiology research team FJAT 4 、 Construction of of avirulent mutants by Tn5 transposon (8) The relative content of EPS of avirulent mutants (8) The relative content of EPS of avirulent mutants

FJAT FAAS Agricultural microbiology research team FJAT 4 、 Construction of of avirulent mutants by Tn5 transposon (9) Pathogencity of avirulent mutents infected in tomato in the 15th day

FJAT FAAS Agricultural microbiology research team FJAT 5 、 New idea of plant vaccin development with avirulent (1) Idea of plant vaccin development with avrirulent RS Tn5 insertion Infect mechanism GPS marked EffiencyInnoculation Plant vaccin production A- RS Field experiment

FJAT FAAS Agricultural microbiology research team FJAT 5 、 New idea of plant vaccin development with avirulent (2) GFP transformation of Tn5-inserted avirulent RS plasmidtransformant morphology of colony Morphology of bacterial body Molecular detection fluorescence microscope

FJAT FAAS Agricultural microbiology research team FJAT 5 、 New idea of plant vaccin development with avirulent (3) Infect mechanism of GFP-marked avirulent RS 24 h 48 h 72 h leaf root stem

FJAT FAAS Agricultural microbiology research team FJAT 5 、 New idea of plant vaccin development with avirulent Effects of a virulent Ralstonia solanacearum strain FJAT-T8 on total POD activity in different parts of tomato T ck T ck T ck Effects of a virulent Ralstonia solanacearum strain FJAT-T8 on total SOD activity in different parts of tomato Effects of a virulent Ralstonia solanacearum strain FJAT-T8 on total PPO activity in different parts of tomato (4) Enzyme activity of tomato seedling Infected with avirulent RS SOD: 25 days POD: 25 days PPO: 25 days Leaf stem root

FJAT FAAS Agricultural microbiology research team FJAT 5 、 New idea of plant vaccin development with avirulent (5) Products of plant vaccin produced with an avirulent RS

FJAT FAAS Agricultural microbiology research team FJAT 5 、 New idea of plant vaccin development with avirulent (6) Protect effiency of bacterial wilt disease by inoculated with an avirulent RS in semi experiments inoculated with an avirulent RS in semi experiments Plant vaccin AVIRULENT was inoculated into tomato seedling in 10 4 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 in 20 days. Plant vaccin Water control Plant vaccin 0% 100% 0% 0% 100% 0% 3days

FJAT FAAS Agricultural microbiology research team FJAT

FAAS Agricultural microbiology research team FJAT 5 、 New idea of plant vaccin development with avirulent CKavirulent avirulent avirulent

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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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