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Invertebrate Microbiology

Insecticidal Bacillus thuringiensis Silences Erwinia carotovora Virulence by a New Form of Microbial Antagonism, Signal Interference

Yi-Hu Dong, Xi-Fen Zhang, Jin-Ling Xu, Lian-Hui Zhang
Yi-Hu Dong
Institute of Molecular and Cell Biology, National University of Singapore, Singapore 117609
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Xi-Fen Zhang
Institute of Molecular and Cell Biology, National University of Singapore, Singapore 117609
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Jin-Ling Xu
Institute of Molecular and Cell Biology, National University of Singapore, Singapore 117609
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Lian-Hui Zhang
Institute of Molecular and Cell Biology, National University of Singapore, Singapore 117609
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  • For correspondence: lianhui@imcb.nus.edu.sg
DOI: 10.1128/AEM.70.2.954-960.2004
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  • FIG. 1.
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    FIG. 1.

    Effect of B. thuringiensis on AHL accumulation and growth of E. carotovora. (A) AHL accumulation during bacterial growth. E. carotovora SCG1 was inoculated alone (▪) or coinoculated, respectively, with B. thuringiensis strain COT1 (*) or B1 (•), E. coli DH5α (▵), or B. fusiformis (□) in LB medium. (B) Time course of bacterial growth. SCG1 (▪), COT1 (⧫), and B1(▴) were incubated and grown separately in LB medium. (C) Cell numbers of SCG1 (▪) and COT1 (⧫) when coinoculated. (D) Cell numbers of SCG1 (▪) and B1 (▴) when coinoculated. The experiment was repeated four times. The mean data are presented.

  • FIG. 2.
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    FIG. 2.

    Effect of B. thuringiensis COT1 on virulence of E. carotovora SCG1. (A) Effect of different treatments on SCG1 infection. C, COT1; W, water; D, E. coli DH5α; Bf, B. fusiformis. The Dip bars represent potato slices pretreated by being dipped into bacterial suspension as described in Materials and Methods. The treated slices were then inoculated with 2.5-μl SCG1 suspensions containing cells equivalent to 5 × 105 or 5 × 104 CFU per site. For the Mix bars, SCG1 was mixed separately with other bacterial culture and inoculated. The final inoculum cell number of SCG1 was equivalent to 2.5 × 105 or 2.5 × 104 CFU, and that for the other bacteria was equivalent to 5 × 104 CFU. The maceration area was measured 20 h after incubation at 28°C. The data were the means of 4 or 12 (COT1) repeats. (B) Development of soft rot symptoms on inoculated potato slices. Potato slices pretreated with COT1 suspension (⧫) or water (▴) were inoculated with 5 μl of SCG1 at a concentration of 2 × 109 CFU/ml. The maceration area was measured at different time points as indicated. The experiment was repeated four times. The mean data are presented.

  • FIG. 3.
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    FIG. 3.

    Soft rot symptoms after treatment with B. thuringiensis COT1. (A) E. carotovora SCG1 infection on potato slices pretreated with COT1 (bottom) or water (top). The slices were inoculated with 2.5 μl of E. carotovora SCG1 suspension containing cells equivalent to 5 × 105, 5 × 104, or 5 × 103 CFU (from left to right). (B) Mix treatment. The cell suspensions of SCG1 at 2 × 108, 2 × 107, or 2 × 106 CFU/ml were mixed separately with equal volumes of water (top) or COT1 suspension cultures at 5 × 108 CFU/ml (bottom). The mixture was inoculated as described above. The final cell numbers of SCG1 inoculated were (from left to right) 2.5 × 105, 2.5 × 104, and 2.5 × 103 CFU. The photographs were taken after incubation for 20 h at 28°C.

  • FIG. 4.
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    FIG. 4.

    Effect of different B. thuringiensis strains on the virulence of E. carotovora. The percentage of maceration sites per inoculation site (A) and the maceration area per macerated site (B) were determined 24 h after inoculation. Potato slices were pretreated by being dipped into water (CK) or the suspensions (5 × 108 CFU/ml) of different subspecies of B. thuringiensis. The slices were inoculated with SCG1 as described in the legend to Fig. 3. The data were recorded from a total of 12 inoculated sites in each treatment.

  • FIG. 5.
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    FIG. 5.

    Effect of B. thuringiensis on in planta colonization of E. carotovora and AHL accumulation. Pretreatment of potato slices with B. thuringiensis B23 suppressed E. carotovora SCG1-GFP infection; no maceration symptoms were visible 24 h after inoculation (A). Fluorescence microscope analysis showed that GFP-expressing E. carotovora cells were confined at the inoculation site (B). Knockout of aiiA in strain B23Δai abolished its AHL-degrading activity (C). The wild-type B23 and B23Δai cell cultures (OD600 = 1.0) were reacted with OHHL in a final concentration of 20 μM for 30 min, and the remaining AHL was determined as described above. Each sample (5 μl) was applied to a single end (marked R) of a separated slice of agar-solidified bioassay medium. Cell suspensions from a fresh culture (OD600 = 0.3) of the AHL biosensor strain 749 were applied as spots at regular increments along the reminder of each slice (marked S). The same amount of OHHL was used as a positive control (CK). The blue dark spots indicate the presence of detectable OHHL that diffused away from the end (R) where the sample was applied; a lack of blue spots indicates the OHHL concentrations fell below the biosensor detection limit.

Tables

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  • TABLE 1.

    Bacterial strains and plasmids used in this study

    Bacterial strain or plasmid and related characteristicsSource or referencea
    Strains
        Bacillus strains
            B. thuringiensis subsp. thuringiensis B1BGSC 4A3
            B. thuringiensis subsp. kurstaki B2BGSC 4D1
            B. thuringiensis subsp. wuhanensis B17, nonflagellarMycogen PSS2A1
            B. thuringiensis B18LC
            B. thuringiensis subsp. kurstaki B22, plasmidlessLC
            B. thuringiensis subsp. israelensis B23, plasmidlessBGSC 4Q7
            B. thuringiensis COT1LC
            B. fusiformis BfLC
            B. thuringiensis subsp. israelensis B23Δai, ΔaiiA::TcThis study
        A. tumefaciens 749 traR tra::lacZ749, AHL indicator29
        E. carotovora
            SCG1, wild-type, virulentLC
            SCG1-GFP, pGEM7-GFP, AmprThis study
        E. coli DH5α F− φ80dlacZΔM15 endA1 hsdR17 (rk− mk−) supE44 thi-1 gyrA96 Δ(lacZYA-argF)33
    Plasmids
        pGEM7 Ampr, cloning vectorPromega
        pGEM-GFP Ampr, GFP expression constructThis study
        pUCTV2 Ampr Tcr, shuttle vector for gene replacement36
        pUCTV2Δai Ampr Tcr, ΔaiiAThis study
    • ↵ a BGSC, Bacillus Genetic Stock Center; LC, laboratory collection.

  • TABLE 2.

    E. carotovora virulence assay on potato slices pretreated with wild-type B. thuringiensis and its mutant lacking AHL-lactonase

    Treatment% of sites with macerationaMaceration area (mm2)b
    5.0 × 105 CFU2.5 × 105 CFU5.0 × 104 CFUDay 1Day 2Day 3
    Water100100100131.1 ± 83.4184.2 ± 72.7195.0 ± 90.4
    B23000000
    B23Δai10010010020.7 ± 13.425.3 ± 11.928.3 ± 13.4
    • ↵ a The pretreated potato slices were inoculated with E. carotovora SCG1 at 5.0 × 105, 2.5 × 105, and 5.0 × 104 CFU. The percentage of inoculation sites showing maceration symptoms was determined 3 days after incubation.

    • ↵ b Maceration areas were measured 1 to 3 days after inoculation with an inoculum of 5.0 × 105 CFU. Data are the means of six replicates.

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Insecticidal Bacillus thuringiensis Silences Erwinia carotovora Virulence by a New Form of Microbial Antagonism, Signal Interference
Yi-Hu Dong, Xi-Fen Zhang, Jin-Ling Xu, Lian-Hui Zhang
Applied and Environmental Microbiology Feb 2004, 70 (2) 954-960; DOI: 10.1128/AEM.70.2.954-960.2004

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Insecticidal Bacillus thuringiensis Silences Erwinia carotovora Virulence by a New Form of Microbial Antagonism, Signal Interference
Yi-Hu Dong, Xi-Fen Zhang, Jin-Ling Xu, Lian-Hui Zhang
Applied and Environmental Microbiology Feb 2004, 70 (2) 954-960; DOI: 10.1128/AEM.70.2.954-960.2004
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KEYWORDS

4-Butyrolactone
Antibiosis
Bacillus thuringiensis
carboxylic ester hydrolases
Pectobacterium carotovorum
Solanum tuberosum

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