Insight into Antifungal Metabolites from Bacillus stercoris 92p Against Banana Cordana Leaf Spot Caused by Neocordana musae

Abstract

Banana crop ranks among the most crucial fruit and food crops in tropical and subtropical areas. Despite advancements in production technology, diseases such as cordana leaf spot, caused by Neocordana musae, remain a significant challenge, reducing productivity and quality. Traditional chemical controls are becoming less effective due to the development of resistance in target pathogens, which pose significant environmental and health concerns. Consequently, there is growing attention toward the development of biocontrol strategies. Here, we identified a new bacterial strain, Bacillus stercoris 92p, from the rhizosphere soil of banana. We evaluated its ability to suppress the growth of N. musae and other fungal pathogens that cause leaf spot disease in bananas. The inhibitory effect of B. stercoris 92p were checked using dual culture assays, microscopic observations, and pot experiments. Furthermore, the biocontrol mechanisms were investigated using whole-genome sequencing and biochemical analyses. The results showed that B. stercoris 92p exhibited significant antifungal activity against N. musae and other fungal pathogens, with inhibition rates exceeding 70%. Microscopic examination revealed significant morphological alterations in the hyphae and conidia of the tested pathogens. In pot experiments, B. stercoris 92p effectively reduced the severity of cordana leaf spot, achieving a biocontrol efficacy of 61.55%. Genomic analysis and biochemical tests indicated that B. stercoris 92p produces various antifungal compounds, including lipopeptides (fengycins and surfactins), hydrolytic enzymes (proteases and amylases), and phosphate-solubilizing metabolites. In conclusion, the study highlights that B. stercoris could potentially be used as a potential biological control agent against cordana leaf spot.

Keywords: Bacillus stercoris; Neocordana musae; antifungal activity; cordana leaf spot of banana; lipopeptide.

Figure 1

(A) Antagonistic activity of Strain 92p against N. musae. The phylogenetic tree of Bacillus sp. Strain 92p was constructed using the maximum-likelihood method for the analysis of two genes: the 16S rRNA gene (B) and the gyrB gene sequence (C).

Figure 2

(A) Antagonistic activity of B. stercoris 92p against phytopathogenic fungi. Antifungal efficacy of B. stercoris 92p against phytopathogenic fungi was evaluated on PDA plates. (B) The graph illustrates the mycelial inhibition (%) by B. stercoris 92p on various phytopathogenic fungi. Bars marked with distinct letters exhibit statistically significant variations (p < 0.05) according to Duncan’s multiple comparison test.

Figure 3

Morphological changes in fungal pathogen hyphae following treatment with B. stercoris 92p. Arrows highlight alterations, including distortions, dissolution, swelling, deformation, beadlike appearance, and vacuolation of structures induced by B. stercoris 92p. Scale bar: 10 μm.

Figure 4

Biocontrol effects of B. stercoris 92p against cordana leaf spot of banana. (A) Banana leaves were treated with sterile water, Strain 92p, and carbendazim, respectively. (B) The lesion area of banana leaves after treatment with ddH₂O, Strain 92p, and carbendazim. Asterisks indicate statistically significant differences according to Duncan’s multiple comparison test (**** p ≤ 0.0001).

Figure 5

Inhibitory effect of B. stercoris 92p cell-free supernatant on N. musae. (A) PDA media were supplemented with varying concentrations (10%, 20%, and 30% v/v) of the 92p sterile supernatant and inoculated with N. musae mycelial discs. A non-supplemented PDA plate served as the control. (B) Bars marked with distinct letters exhibit statistically significant variations (p ≤ 0.05) according to Duncan’s multiple comparison test.

Figure 6

The enzymatic activity and the secondary metabolites produced by B. stercoris Strain 92p. (A) Protease production, (B) Amylase production, (C) Phosphate solubilization assay, (D) Cellulase production, (E) β-1,3-Glucanase production, (F) Siderophore production.

Figure 7

Circular map of B. stercoris 92p genome. The outermost circle visually represents the size and scale of the genome. The default circles from outer to inner correspond to genome size markers, gene information on the forward and reverse strand, non-coding RNA, repetitive elements, GC content, and GC-Skew.

Figure 8

The heatmap displays the average nucleotide identity among strains 92p and 15 Bacillus. ANI values were computed using FastANI for pairwise genome comparisons, and the heatmap illustrates the percentage of ANI among 15 Bacillus strains, with higher values represented by reddish colors to distinguish strains of the same species.

Figure 9

MS/MS spectra of surfactin ions. (A): m/z 1008.6574, (B): m/z 1022.6784, (C): m/z 1036.5731.

Figure 10

The MS/MS analysis of four fengycins (A–D). (A) m/z 1463.8242, (B) m/z 1477.8224, (C): m/z 1491.8338, (D) m/z 1505.8638. The black box labeled fingerprint product ions 1080 and 966 indicated that the m/z values 1463.8242 and 1477.8224 correspond to fengycin A. The black box labeled fingerprint product ions 1108 and 994 indicated that the m/z values 1491.8338 and 1505.8638 correspond to fengycin B.