Fig 1.
B. gladioli NB6 and its culture filtrate protect rice seedlings from infection by B. glumae and B. plantarii.
Rice seeds were co-inoculated with pathogens (Bg: B. glumae, Bp: B. plantarii) and tested biocontrol bacteria (NB6: B. gladioli NB6, M1064: B. gladioli MAFF301064), their respective culture filtrates (CF), or sterilized distilled water (SDW). (A) Disease symptoms in rice seedlings resulting from the inoculated seeds at 8 days postinoculation (dpi). (B and C) Box plots showing the shoot length of rice seedlings at 8 dpi: the central lines indicate the medians, the box limits represent the upper and lower quartiles, and the whiskers extend to the maximum and minimum values. Different letters indicate statistically significant differences between groups (Tukey–Kramer test, P < 0.05, n = 9). The relative biomass of Bg (D) and Bp (E) in rice seedlings at 8 dpi was measured by quantitative PCR. Two seedlings were used for total DNA extraction. Data are presented as means ± SE of log10-transformed values + 1, relative to the plants inoculated with pathogens only. Values are indicated above the bars, and “BDL” signifies below detection limit. Different letters indicate statistically significant differences between groups (Tukey–Kramer test, P <0.05, n = 4). These experiments were repeated three times, and similar results were obtained. Figures show the results of one representative experiment.
Fig 2.
Purification of the plant-protective substance contained in the culture filtrate of B. gladioli NB6.
(A) Schematic representation of the purification and identification processes for proteins contained in the culture filtrate (CF) of B. gladioli NB6, which were conducted using a transposon insertion mutant of NB6, NB6-M17. (B) Twenty μl of a gel filtration fraction exhibiting protective activity against B. glumae (Bg) was analyzed via SDS-PAGE. Arrows indicate four visible bands that were subjected to mass spectrometry analysis. (C) Summary of the results of mass spectrometry analysis. (D and E) Rice seeds were co-inoculated with Bg and NB6, along with its two independent gene-disruptive mutants (ΔFII #3 and #8, Δcap #4 and #11, and ΔporA #6 and #8), their respective culture filtrates (CFs), or sterilized distilled water (SDW). Box plots show the shoot length of rice seedlings at 7 days postinoculation: the central lines indicate the medians, the box limits represent the upper and lower quartiles, and the whiskers extend to the maximum and minimum values. Different letters indicate statistically significant differences between groups (Tukey–Kramer test, P < 0.05, n = 10). Experiments were repeated twice, and similar results were obtained. Figures show the results of one representative experiment.
Fig 3.
Identification of a prophage in the B. gladioli NB6 genome encoding a plant-protective substance.
(A) Schematic representation of the genetic organization of Region 1 and Region 2 prophages in the B. gladioli NB6 genome. Each gene is indicated by arrows with different colors based on their annotations, i.e., white for hypothetical proteins, cyan for phage-related proteins, blue for phage head proteins, and yellow for proteins identified by mass spectrometry. Chords with black–white gradation between regions represent protein similarities. (B) Rice seeds were co-inoculated with B. glumae (Bg) and NB6, along with its two independent gene-disruptive mutants (Δ1440–1500 #4 and #5), their respective culture filtrates (CFs), or sterilized distilled water (SDW). The box plot shows the shoot length of rice seedlings at 8 days postinoculation (dpi): the central lines indicate the medians, the box limits represent the upper and lower quartiles, and the whiskers extend to the maximum and minimum values. Different letters indicate statistically significant differences between groups (Tukey–Kramer test, P < 0.05, n = 10). The experiment was repeated twice, and similar results were obtained. (C) The recombinant FII protein (rFII) was produced using Escherichia coli. The monomeric rFII was purified and resolved via SDS-PAGE. (D) Rice seeds were co-inoculated with Bg and the CF of NB6, rFII, or 50 mM Tris-HCl buffer (pH 7.5). The CF and rFII protein concentrations were adjusted to 0.2 mg/ml. The box plot shows the shoot length of rice seedlings at 8 dpi and has the same format as that in (B). Different letters indicate statistically significant differences between groups (Tukey–Kramer test, P < 0.05, n = 8). The experiment was repeated twice, and similar results were obtained. Figures show the results of one representative experiment.
Fig 4.
Identification of the tailocin BglaTNB6 produced by B. gladioli with antibacterial activity against B. glumae and B. plantarii.
(A) Rice seeds were co-inoculated with B. glumae (Bg) and either sterilized distilled water (SDW) or the precipitate and supernatant obtained from ultracentrifugation of the culture filtrate of B. gladioli NB6. The protein concentrations of the precipitate and supernatant were adjusted to 1.0 mg/ml. The box plot shows the shoot length of rice seedlings at 8 days postinoculation: the central lines indicate the medians, the box limits represent the upper and lower quartiles, and the whiskers extend to the maximum and minimum values. Different letters indicate statistically significant differences between groups (Tukey–Kramer test, P < 0.05, n = 10). The inoculation test was repeated twice, and similar results were obtained. (B-E) Structures recognized as tailocins (B) and phages (C-E) observed in the precipitate of NB6 via transmission electron microscopy. Scale bars indicate 100 nm. Magnification, ×50,000. (F) Antibacterial assay of the NB6 precipitate. Five-μl serial dilutions (1/5 stepwise) of the precipitate were spotted onto LB medium containing Bg (Left) and B. plantarii (Bp, right) and incubated overnight to evaluate inhibition zones. (G) Antibacterial assay of the precipitate of NB6 and its gene-disruptive mutants. Five μl of the precipitate or 0.5 mg/ml kanamycin (Km) were spotted onto LB medium containing Bg (left) and Bp (right) and incubated overnight to evaluate inhibition zones. These antibacterial assays were repeated twice, and similar results were obtained. Figures show the results of one representative experiment.
Fig 5.
BglaTNB6 greatly contributed for the biocontrol activity of B. gladioli NB6 against B. plantarii.
Rice seeds were co-inoculated with B. plantarii (Bp) and B. gladioli NB6, along with its two independent gene-disruptive mutants (ΔFII #3 and #8), their respective culture filtrates (CFs), or sterilized distilled water (SDW). (A) Disease symptoms in rice seedlings resulting from the inoculated seeds at 7 days post inoculation (dpi). (B) The box plot shows the shoot length of rice seedlings at 7 dpi: the central lines indicate the medians, the box limits represent the upper and lower quartiles, and the whiskers extend to the maximum and minimum values. Different letters indicate statistically significant differences between groups (Tukey–Kramer test, P < 0.05, n = 10). The experiment was repeated three times, and similar results were obtained. Figure shows the results of one representative experiment.