Bacillus subtilis ED24 Controls Fusarium culmorum in Wheat Through Bioactive Metabolite Secretion and Modulation of Rhizosphere Microbiome

Abstract

Fusarium culmorum is a soil-borne fungal pathogen causing root and stem rot, seedling blight, and significant yield losses in small grain cereals, including wheat. This study aimed to evaluate the antifungal potential of Bacillus subtilis ED24, an endophytic strain isolated from Ziziphus lotus (L.) roots, and its effects on wheat growth and yield under controlled conditions. In vitro assays demonstrated that B. subtilis ED24 inhibited F. culmorum mycelial growth by up to 87%, associated with the secretion of 37 distinct secondary metabolites, predominantly involved in carbon cycling. In pot experiments, B. subtilis ED24 significantly enhanced wheat germination (85%) and growth compared to infected plants treated with the chemical fungicide tebuconazole. Although nutrient analysis showed significantly higher shoot nitrogen (32.34 mg/pot) and phosphorus (2.41 mg/pot) contents in the B. subtilis ED24 treatment compared to tebuconazole (8.11 and 0.18 mg/pot, respectively), no significant differences were observed when compared to the infected control (C-). Similarly, B. subtilis ED24 led to improved thousand grain weight (40.4 g), protein content (19.98%), and ash content (1.95%) relative to tebuconazole (29.1 g, 18.31%, and 1.74%, respectively), yet these values did not differ significantly from the infected control (C-). Notably, the number of seeds per pot was significantly increased by B. subtilis ED24 compared to the infected control (C-) (113.8 seeds/pot vs. 54.2 seeds/pot). Additionally, B. subtilis ED24 modulated the wheat rhizosphere microbiome, enriching beneficial taxa such as Eurotiomycetes fungal class and the bacterial genus Paramesorhizobium. These findings suggest that the antifungal activity and growth-promoting effects of B. subtilis ED24 are likely mediated through the synthesis of unique bioactive metabolites and microbiome modulation, offering a promising sustainable alternative to chemical fungicides in wheat production.

Keywords: Triticum durum; Ziziphus lotus (L.) Desf.; Endophyte; Propionic acid; Rhizosphere microbiota; Secondary metabolites; Tebuconazole.

Fig. 1

A Effect of B. subtilis ED24 on mycelial growth of the fungi after 5 days of incubation. Vertical bars represent means and standard errors (mean ± SE) based on two replicates. Distinct lowercase letters indicate statistical significance at p ≤ 0.05 between each treatment and its control. B Effect of B. subtilis ED24 on F. culmorum growth after 5 days of incubation

Fig. 2

Comparative visualization of microbial metabolite profiles. A Intersection analysis of shared and unique metabolites across treatments, B heatmap of relative abundances of metabolites across treatments, and C relative abundance of compounds across treatments

Fig. 3

Effect of B. subtilis ED24 and TEBU treatments on wheat seed germination rate measured 7 days after sowing and their vigor index. Vertical bars depict the means and standard errors (mean ± SE) based on five replicates. Distinct lowercase letters indicate statistical significance at p ≤ 0.05

Fig. 4

Effect of B. subtilis ED24 and TEBU treatments on the shoot nitrogen, phosphorus, and potassium contents. Vertical bars depict the means and standard errors (Mean ± SE) based on five replicates, and each replicate consists of five wheat plants per pot. Distinct lowercase letters indicate statistical significance at p ≤ 0.05

Fig. 5

Effect of B. subtilis ED24 and TEBU treatments on wheat root morphology and F. culmorum spore colonization. A Wheat root length and surface area under B. subtilis ED24 and TEBU treatments. B Root system architecture of wheat plants treated with B. subtilis ED24 and TEBU, scanned, and analyzed using WinRHIZO. C Microscopic observations of F. culmorum spores under B. subtilis ED24 and TEBU treatments; the presence of spores is highlighted with orange arrows

Fig. 6

Relative abundance and NMDS analysis of wheat rhizospheric bacteria and fungi communities in infected control group (C-), B. subtilis ED24, and TEBU treatments

Fig. 7

Heatmap displays the relative abundance of the 100 most frequent amplicon sequence variants in the bacterial and fungal communities of the wheat rhizosphere of the infected control group (C-), B. subtilis ED24, and TEBU treatments

Fig. 8

Heat stress illustrating the wheat rhizospheric bacteria and fungi distribution under B. subtilis ED24 and TEBU treatments compared to the infected control group (C-)

Fig. 9

Principal component analysis (PCA) illustrating wheat rhizospheric bacteria and fungi distribution along with plant growth parameters under B. subtilis ED24 and TEBU treatments