Killing Effect of Bacillus Velezensis FZB42 on a Xanthomonas Campestris pv. Campestris (Xcc) Strain Newly Isolated from Cabbage Brassica Oleracea Convar. Capitata (L.): A Metabolomic Study

Abstract

The potential use of Bacillus velezensis FZB42 for biological control of various phytopathogens has been documented over the past few years, but its antagonistic interactions with xanthomonads has not been studied in detail. Novel aspects in this study consist of close observation of the death of Xanthomonas campestris pv. campestris cells in a co-culture with B. velezensis FZB42, and quantification of lipopeptides and a siderophore, bacillibactin, involved in the killing process. A new robust Xcc-SU isolate tolerating high concentrations of ferric ions was used. In a co-culture with the antagonist, the population of Xcc-SU was entirely destroyed within 24-48 h, depending on the number of antagonist cells used for inoculation. No inhibitory effect of Xcc-SU on B. velezensis was observed. Bacillibactin and lipopeptides (surfactin, fengycin, and bacillomycin) were present in the co-culture and the monoculture of B. velezensis. Except for bacillibactin, the maximum contents of lipopeptides were higher in the antagonist monoculture compared with the co-culture. Scanning electron microscopy showed that the death of Xcc-SU bacteria in co-culture was caused by cell lysis, leading to an enhanced occurrence of distorted cells and cell ghosts. Analysis by mass spectrometry showed four significant compounds, bacillibactin, surfactin, fengycin, and bacillomycin D amongst a total of 24 different forms detected in the co-culture supernatant: Different forms of surfactin and fengycin with variations in their side-chain length were also detected. These results demonstrate the ability of B. velezensis FZB42 to act as a potent antagonistic strain against Xcc.

Keywords: Bacillus velezensis FZB42; Xanthomonas campestris pv. campestris; antagonism; cyclic lipopeptides; killing effect; metabolomic analysis; siderophore.

Figure 1

Inhibition of growth of Xcc strains by B. velezensis FZB42 on BH agar medium at 8 μM Fe³⁺ (left column) and 200 μM Fe³⁺ (right column): (A) top, Xcc 1279A; bottom, Xcc SU; (B) top, Xcc 3811; bottom, Xcc 3871A.

Figure 2

Xcc-SU dying rates in dual cultures with B. velezensis FZB42 grown in liquid medium. The ratios of B. velezensis to X. campestris SU at the time of inoculation was 1:3 (B), 1:15 (C), and 1:100 (D) in CFU/mL. (A) Representative growth curves of both microorganisms in control monocultures. The individual values in the graphs show the arithmetic means of CFU/mL measured at a given time in two independent cultures. The open dots in the Xcc plots indicate that no living Xcc bacteria were detected in the dual culture. R Core Team (2021) [46] free software environment for statistical computing and graphics was used for graphical presentation of the data. B. velezensis FZB42, blue line; Xcc-SU, red line.

Figure 3

Production of biocontrol metabolites in a dual culture of B. velezensis FZB42 and Xcc-SU grown in liquid medium. The ratio of B. velezensis to Xcc-SU at the time of inoculation was 1:100 (CFU/mL). Dual culture, full columns; B. velezensis monoculture, empty columns.

Figure 4

Time course of the interaction of B. velezensis FZB42 and Xcc-SU growing in a dual culture visualized by SEM (scale 2 μm): (A) start inoculation: Xcc-SU monoculture, (B) start inoculation: B. velezensis monoculture, (C) 48 h after inoculation: Xcc-SU monoculture, (D) 48 h after inoculation: B. velezensis monoculture, (E) 12 h after inoculation: dual culture, (F) 48 h after inoculation: dual culture. The ratio of B. velezensis cells to Xcc-SU cells at the time of inoculation was 1:100.